Treatment of Cancer Using Inhibitors of TGF-Beta and PD-1

ABSTRACT

The present disclosure relates, in general, to combination therapy using an inhibitor of transforming growth factor beta (TGFβ) and an inhibitor of programmed cell death protein 1 (PD-1) for treating cancer or preventing recurrence of cancer diseases such as lung cancer, prostate cancer, breast cancer, hepatocellular cancer, esophageal cancer, colorectal cancer, pancreatic cancer, bladder cancer, kidney cancer, ovarian cancer, stomach cancer, fibrotic cancer, glioma and melanoma, and metastases thereof.

This application claims the priority benefit of U.S. Provisional PatentApplication No. 62/143,016, filed Apr. 3, 2015 and U.S. ProvisionalPatent Application No. 62/191,797, filed Jul. 13, 2015, hereinincorporated by reference in their entirety.

This invention was made with government support under Grant NumberR21CA164772 and U01CA084244 awarded by the National Institutes ofHealth. The government has certain rights in the invention.

INCORPORATION BY REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY

Incorporated by reference in its entirety is a computer-readablenucleotide/amino acidsequence listing submitted concurrently herewithand identified as follows: One 17,107 byte ASCII (Text) file named“49343_SeqListing.txt” created on Apr. 1, 2016.

FIELD OF THE INVENTION

The present disclosure relates, in general, to combination therapy fortreating cancer or preventing the recurrence of cancer comprisingadministering to a subject in need thereof a therapeutically effectiveamount of an inhibitor of transforming growth factor beta (TGFβ) and aninhibitor of programmed cell death protein 1 (PD-1).

BACKGROUND

Cancer immunotherapy refers to methods of activating the immune systemto induce tumor regression and disease stabilization (Mellman I et al.,Nature. 480, 7378: 480-9 (2011)). Antibody therapy directed againstcertain negative immunologic regulators (immune checkpoints) has beenshown to be successful as an anti-tumor treatment in several cancertypes (Postow et al., J Clin Oncol 33: 9, (2015)).

The transforming growth factor beta (TGFβ) protein family consists ofthree distinct isoforms found in mammals (TGFβ1, TGβ2, and TGβ3). TheTGFβ proteins activate and regulate multiple gene responses thatinfluence disease states, including cell proliferative, inflammatory,and cardiovascular conditions. TGFβ is a multifunctional cytokineoriginally named for its ability to transform normal fibroblasts tocells capable of anchorage-independent growth. The TGFβ molecules areproduced primarily by hematopoietic and tumor cells and can regulate,i.e., stimulate or inhibit, the growth and differentiation of cells froma variety of both normal and neoplastic tissue origins (Sporn et al.,Science, 233: 532 (1986)), and stimulate the formation and expansion ofvarious stromal cells.

The TGFβs are known to be involved in many proliferative andnon-proliferative cellular processes such as cell proliferation anddifferentiation, embryonic development, extracellular matrix formation,bone development, wound healing, hematopoiesis, and immune andinflammatory responses. See e.g., Pircher et al, Biochem. Biophys. Res.Commun., 136: 30-37 (1986); Wakefield et al., Growth Factors, 1: 203-218(1989); Roberts and Sporn, pp 419-472 in Handbook of ExperimentalPharmacology eds M. B. Sporn & A. B. Roberts (Springer, Heidelberg,1990); Massague et al., Annual Rev. Cell Biol., 6: 597-646 (1990);Singer and Clark, New Eng. J. Med., 341: 738-745 (1999). Also, TGFβ isused in the treatment and prevention of diseases of the intestinalmucosa (WO 2001/24813). TGFβ is also known to have strongimmunosuppressive effects on various immunologic cell types, includingcytotoxic T lymphocyte (CTL) inhibition (Ranges et al., J. Exp. Med.,166: 991, 1987), Espevik et al., J. Immunol., 140: 2312, 1988),depressed B cell lymphopoiesis and kappa light-chain expression (Lee etal., J. Exp. Med., 166: 1290, 1987), negative regulation ofhematopoiesis (Sing et al., Blood, 72: 1504, 1988), down-regulation ofHLA-DR expression on tumor cells (Czarniecki et al., J. Immunol., 140:4217, 1988), and inhibition of the proliferation of antigen-activated Blymphocytes in response to B-cell growth factor (Petit-Koskas et al.,Eur. J. Immunol., 18: 111, 1988). See also U.S. Pat. No. 7,527,791.

TGFβ isoform expression in cancer is complex and variable with differentcombinations of TGFβ isoforms having different roles in particularcancers. TGFβ molecules can act both as tumor suppressors and tumorpromoters. For example, deletion or dowregulation of TGFβ signaling inanimals can result in increased breast cancer, intestinal cancer,pancreatic cancer, colon cancer and squamous cell carcinoma, indicatingthe presence of TGFβ is important to prevent or slow tumor progression(Yang et al., Trends Immunol 31:220-27, 2010). However, overexpressionof TGFβ is known to be pro-oncogenic and increased expression isdetected in many tumor types (Yang et al., supra).

Antibodies to TGFβ have been described in U.S. Pat. Nos. 7,527,791;7,927,593; 7,494,651; 7,369,111; 7,151,169; 6,492,497; 6,419,928;6,090,383; 5,783,185; 5,772,998; 5,571,714; and 7,723,486 and 8,569,462.

Programmed cell death protein 1 (PD-1), also known as cluster ofdifferentiation 279 (CD279), is a cell surface co-inhibitory receptorexpressed on activated T cells, B cells and macrophages, and is acomponent of immune checkpoint blockade (Shinohara et al., Genomics.,23(3):704, (1994); Francisco et al., Immunol Rev., 236: 219, (2010)).PD-1 limits the activity of T cells upon interaction with its twoligands PD-L1 (also known as B7-H1; CD274) and PD-L2 (B7-DC; CD273)(Postow et al., J Clin Oncol., 33: 9, (2015)). Interaction of PD-1 withPD-L1 and PD-L2, reduces T-cell proliferation, cytokine production, andcytotoxic activity (Freeman G J et al., J Exp Med., 192:1027-34, (2000);Brown J A et al., J Immunol., 170:1257-66, (2003)).

Recently, two monoclonal antibodies have been approved by the U.S. Foodand Drug Administration (FDA) for the inhibition of PD-1 immunotherapy.Pembrolizumab (KEYTRUDA®, Merck Sharp & Dohme Corp.) is approved for usein metastatic melanoma, and nivolumab (Opdivo®, Bristol-Myers Squibb) isapproved for use in metastatic melanoma and metastatic squamousnon-small cell lung cancer (NSCLC). Both of these antibodies bind to thePD-1 receptor and block its interaction with its ligands, PD-L1 andPD-L2.

Inhibitors of PD-L1 have also been shown to be effective at inhibitingsolid tumors in bladder cancer, head and neck cancer, andgastrointestinal cancers (Herbst R S et al., J Clin Oncol., 31: 3000(2013); Heery C R et al., J Clin Oncol., 32: 5s, 3064 (2014); Powles Tet al., J Clin Oncol, 32: 5s, 5011(2014); Segal N H et al., J ClinOncol., 32: 5s, 3002 (2014)).

Antibodies to PD-1 have been described in U.S. Pat. Nos. 8,735,553;8,617,546; 8,008,449; 8,741,295; 8,552,154; 8,354,509; 8,779,105;7,563,869; 8,287,856; 8,927,697; 8,088,905; 7,595,048; 8,168,179;6,808,710; 7,943,743; 8,246,955; and 8,217,149.

In the setting of cancer, multiple mechanisms of immune suppression mayprevent immunotherapy from being effective. In some cases tumors arerefractory to mono-immunotherapy and only a minor fraction of cancersfully respond. Therefore the use of combinations of immunotherapeuticagents will likely be required for optimal patient responses (Hodi F Set al., Adv Immunol., 90:341-68, 2006; Postow et al., J Clin Oncol., 33:9, 2015).

Recently TGFβ inhibition combined with inhibition of immune checkpointprotein CTLA-4 has been demonstrated to be effective at suppressingmelanoma tumor growth and metastasis (Hanks et al., J Clin Oncol 32: 5s,2014). Combinational immunotherapy approaches using inhibitors ofPD-1/PD-L1 and CTLA-4 are currently being evaluated (Sznol M et al., JClin Oncol., 32: 5s, 2014; Wolchok J D et al., N Engl J Med., 369:122-133, 2013; Callahan et al., J Clin Oncol., 32:5s, 2014 and reviewedin Postow et al., J Clin Oncol., 33: 9, (2015)). Studies have describedsynergistic upregulation of IFNγ in effector T cells from tumor-draininglymph nodes following simultaneous blockade of PD-L1 and TGFβ using acombination of monoclonal antibodies implicating PD-L1 and TGFβ insuppressing cellular responses to active immunization in thetumor-bearing host (Wei et al., Cancer Res, 68: 13, 2008).

SUMMARY OF THE INVENTION

The present disclosure relates, in general, to materials and methods fortreating cancer or preventing the recurrence of cancer using inhibitorsof TGFβ and PD-1 in combinational therapy. Inhibition of TGFβ has beendemonstrated to stimulate chemokine secretion and inflammation, whilePD-1 blockade has been shown to suppress immune inhibitory mechanisms.The data presented herein demonstrate that inhibitors of TGFβ, inparticular when administrated with inhibitors of PD-1, elicit tumorregression in mouse models of cancer.

In various embodiments, the present disclosure provides a method fortreating cancer or preventing the recurrence of cancer comprisingadministering to a subject in need thereof therapeutically effectiveamounts of an inhibitor of transforming growth factor beta (TGFβ) and aninhibitor of Programmed cell death protein 1 (PD-1).

In various embodiments, the methods contemplate use of an antibody thatbinds transforming growth factor beta (TGFβ)1, TGFβ2 and TGβ3. In someembodiments, the antibody neutralizes activity of TGFβ1 and TGFβ2 to agreater extent than TGβ3. In some embodiments, antibody neutralizationof TGFβ1 and TGFβ2 is at least 2-50 fold, 10-100 fold, 2-fold, 5-fold,10-fold, 25-fold, 50-fold or 100-fold, or 20-50%, 50-100%, 20%, 25%,30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% more potent thatneutralization of TGβ3. Exemplary neutralization assays contemplatedherein include, but are not limited to, an interleukin-11 release assayand an HT-2 cell proliferation assay. In addition, a TGFβ activity assaycan be carried out to determine if an antibody disclosed herein inhibitsone TGFβ isoform preferentially, including a pSMAD phosphorylation assayor an rhLAP binding assay. In a further embodiment, the antibody has alower IC50 (i.e., better binding, greater potency) for TGFβ1 and TGFβ2compared to TGFβ.

In various embodiments, the methods contemplate use of an antibody thatbinds TGFβ, TGFβ2 and TGFβ3 comprising: a heavy chain complementarydetermining repeat (CDR), CDR1 amino acid sequence set forth in SEQ IDNOs: 13, 19 and 25, or a variant thereof having at least 85% identitythereto; a heavy chain CDR2 amino acid sequence set forth in SEQ ID NOs:14, 20 and 26, or a variant thereof having at least 85% identitythereto; a heavy chain CDR3 amino acid sequence set forth in SEQ ID NOs:15, 21 and 27, or a variant thereof having at least 85% identitythereto; a light chain CDR1 amino acid sequence set forth in SEQ ID NOs:16, 22 and 28, or a variant thereof having at least 85% identitythereto; a light chain CDR2 amino acid sequence set forth in SEQ ID NOs:17, 23 and 29, or a variant thereof having at least 85% identitythereto; and a light chain CDR3 amino acid sequence set forth in SEQ IDNOs: 18, 24 and 30, or a variant thereof having at least 85% identitythereto. In some embodiments, it is contemplated that an antibody usefulin the methods comprises an amino acid sequence at least 85% identicalto a heavy chain variable region amino acid sequence set forth in SEQ IDNOs: 2, 6 and 10. In a related embodiment, the antibody comprises anamino acid sequence at least 95% identical to a heavy chain variableregion amino acid sequence set forth in SEQ ID NOs: 2, 6 and 10.

In a related embodiment, the antibody comprises an amino acid sequenceat least 85% identical to a light chain variable region amino acidsequence set forth in SEQ ID NOs: 4, 8 and 12. In a further embodiment,the antibody comprises an amino acid sequence at least 95% identical toa light chain variable region amino acid sequence set forth in SEQ IDNOs: 4, 8 and 12. In still another embodiment, the antibody comprises alight chain variable region amino acid sequence set forth in SEQ ID NOs:4, 8 and 12.

In various embodiments, the methods contemplate use of an antibody thatbinds to TGFβ1 and TGFβ2 with greater affinity than to TGFβ3 comprising:a heavy chain CDR1 amino acid sequence set forth in SEQ ID NO: 19, or avariant thereof in which one or two amino acids have been changed; aheavy chain CDR2 amino acid sequence set forth in SEQ ID NO: 20, or avariant thereof in which one or two amino acids have been changed; aheavy chain CDR3 amino acid sequence set forth in SEQ ID NO: 21, or avariant thereof in which one or two amino acids have been changed; alight chain CDR1 amino acid sequence set forth in SEQ ID NO: 22, or avariant thereof in which one or two amino acids have been changed; alight chain CDR2 amino acid sequence set forth in SEQ ID NO: 23, or avariant thereof in which one or two amino acids have been changed; and alight chain CDR3 amino acid sequence set forth in SEQ ID NO: 24, or avariant thereof in which one or two amino acids have been changed.

In various embodiments, the methods contemplate use of an antibody thatneutralizes activity of TGFβ1 and TGFβ2 to a greater extent than itneutralizes the activity of TGFβ3 comprising: a heavy chain CDR1 aminoacid sequence set forth in SEQ ID NO: 19, or a variant thereof in whichone or two amino acids have been changed; a heavy chain CDR2 amino acidsequence set forth in SEQ ID NO: 20, or a variant thereof in which oneor two amino acids have been changed; a heavy chain CDR3 amino acidsequence set forth in SEQ ID NO: 21, or a variant thereof in which oneor two amino acids have been changed; a light chain CDR1 amino acidsequence set forth in SEQ ID NO: 22, or a variant thereof in which oneor two amino acids have been changed; a light chain CDR2 amino acidsequence set forth in SEQ ID NO: 23, or a variant thereof in which oneor two amino acids have been changed; and a light chain CDR3 amino acidsequence set forth in SEQ ID NO: 24, or a variant thereof in which oneor two amino acids have been changed.

In one aspect, the methods contemplate use of an antibody that bindsTGFβ1, TGFβ2 and TGFβ3 comprising: (a) a heavy chain CDR1 amino acidsequence set forth in SEQ ID NO: 25, or a variant thereof in which oneor two amino acids have been changed; (b) a heavy chain CDR2 amino acidsequence set forth in SEQ ID NO: 26, or a variant thereof in which oneor two amino acids have been changed; (c) a heavy chain CDR3 amino acidsequence set forth in SEQ ID NO: 27, or a variant thereof in which oneor two amino acids have been changed; (d) a light chain CDR1 amino acidsequence set forth in SEQ ID NO: 28, or a variant thereof in which oneor two amino acids have been changed; (e) a light chain CDR2 amino acidsequence set forth in SEQ ID NO: 29, or a variant thereof in which oneor two amino acids have been changed; and (f) a light chain CDR3 aminoacid sequence set forth in SEQ ID NO: 30, or a variant thereof in whichone or two amino acids have been changed.

In another aspect, an antibody described herein comprises (a) a heavychain CDR1 amino acid sequence set forth in SEQ ID NO: 13, or a variantthereof in which one or two amino acids have been changed; (b) a heavychain CDR2 amino acid sequence set forth in SEQ ID NO: 14, or a variantthereof in which one or two amino acids have been changed; (c) a heavychain CDR3 amino acid sequence set forth in SEQ ID NO: 15, or a variantthereof in which one or two amino acids have been changed; (d) a lightchain CDR1 amino acid sequence set forth in SEQ ID NO: 16, or a variantthereof in which one or two amino acids have been changed; (e) a lightchain CDR2 amino acid sequence set forth in SEQ ID NO: 17, or a variantthereof in which one or two amino acids have been changed; and (f) alight chain CDR3 amino acid sequence set forth in SEQ ID NO: 18, or avariant thereof in which one or two amino acids have been changed.

In various embodiments, a TGFβ inhibitor useful in the methods is anantibody comprising: a heavy chain CDR1 amino acid sequence set forth inSEQ ID NO: 19, or a variant thereof in which one or two amino acids havebeen changed; a heavy chain CDR2 amino acid sequence set forth in SEQ IDNO: 20, or a variant thereof in which one or two amino acids have beenchanged; a heavy chain CDR3 amino acid sequence set forth in SEQ ID NO:21, or a variant thereof in which one or two amino acids have beenchanged; a light chain CDR1 amino acid sequence set forth in SEQ ID NO:22, or a variant thereof in which one or two amino acids have beenchanged; a light chain CDR2 amino acid sequence set forth in SEQ ID NO:23, or a variant thereof in which one or two amino acids have beenchanged; and a light chain CDR3 amino acid sequence set forth in SEQ IDNO: 24, or a variant thereof in which one or two amino acids have beenchanged.

In a related embodiment, an antibody described herein comprises theheavy chain variable region amino acid sequence as set forth in SEQ IDNO: 10 and the light chain variable region amino acid sequence as setforth in SEQ ID NO: 12.

In a related embodiment, an antibody described herein comprises theheavy chain variable region amino acid sequence is set forth in SEQ IDNO: 2 and the light chain variable region amino acid sequence is setforth in SEQ ID NO: 4.

In a related embodiment, an antibody described herein comprises theheavy chain variable region amino acid sequence is set forth in SEQ IDNO: 6 and the light chain variable region amino acid sequence is setforth in SEQ ID NO: 8.

In some embodiments, an antibody useful in the methods further comprisesa heavy chain constant region, wherein the heavy chain constant regionis a modified or unmodified IgG, IgM, IgA, IgD, IgE, a fragment thereof,or combinations thereof.

In one embodiment, an antibody used herein further comprises a humanlight chain constant region attached to said light chain variableregion. In some embodiments, the light chain constant region is amodified or unmodified lambda light chain constant region, a kappa lightchain constant region, a fragment thereof, or combinations thereof.

In some embodiments, it is contemplated that the PD-1 inhibitor is anantibody that binds PD-1 such as a monoclonal antibody disclosed in theDetailed Description. In various embodiments, the anti-PD-1 antibodyinhibits or blocks binding of the PD-1 receptor to one or both of itsligands, PD-L1 and PD-L2.

In a related aspect, the disclosure describes the use of a PD-1inhibitor used in combination with one or more of the above TGFβmonoclonal antibodies.

In a related aspect, the disclosure provides a method for treatingcancer or preventing the reoccurrence of cancer comprising administeringto a subject in need thereof a therapeutically effective amount of theantibodies or pharmaceutical compositions contemplated herein. Incertain embodiments, the cancer is selected from the group consisting ofesophageal cancer, pancreatic cancer, metastatic pancreatic cancer,metastatic adenocarcinoma of the pancreas, bladder cancer, stomachcancer, fibrotic cancer, glioma, malignant glioma, diffuse intrinsicpontine glioma, recurrent childhood brain neoplasm renal cell carcinoma,clear-cell metastatic renal cell carcinoma, kidney cancer, prostatecancer, metastatic castration resistant prostate cancer, stage IVprostate cancer, metastatic melanoma, melanoma, malignant melanoma,recurrent melanoma of the skin, melanoma brain metastases, stage IIIAskin melanoma; stage IIIB skin melanoma, stage IIIC skin melanoma; stageIV skin melanoma, malignant melanoma of head and neck, lung cancer, nonsmall cell lung cancer (NSCLC), squamous cell non-small cell lungcancer, breast cancer, recurrent metastatic breast cancer,hepatocellular carcinoma, hodgkin's lymphoma, follicular lymphoma,non-hodgkin's lymphoma, advanced B-cell NHL, HL including diffuse largeB-cell lymphoma (DLBCL) including DLBCL following autologous stem celltransplantation, multiple myeloma, chronic myeloid leukemia, adult acutemyeloid leukemia in remission; adult acute myeloid leukemia withInv(16)(p13.1q22); CBFB-MYH11; adult acute myeloid leukemia witht(16;16)(p13.1;q22); CBFB-MYH11; adult acute myeloid leukemia witht(8;21)(q22;q22); RUNX1-RUNX1T1; adult acute myeloid leukemia witht(9;11)(p22;q23); MLLT3-MLL; adult acute promyelocytic leukemia witht(15;17)(q22;q12); PML-RARA; alkylating agent-related acute myeloidleukemia, chronic lymphocytic leukemia, richter's syndrome; waldenstrommacroglobulinemia, adult glioblastoma; adult gliosarcoma, recurrentglioblastoma, recurrent childhood rhabdomyosarcoma, recurrent ewingsarcoma/peripheral primitive neuroectodermal tumor, recurrentneuroblastoma; recurrent osteo sarcoma, colorectal cancer, MSI positivecolorectal cancer; MSI negative colorectal cancer, nasopharyngealnonkeratinizing carcinoma; recurrent nasopharyngeal undifferentiatedcarcinoma, cervical adenocarcinoma; cervical adenosquamous carcinoma;cervical squamous cell carcinoma; recurrent cervical carcinoma; stageIVA cervical cancer; stage IVB cervical cancer, anal canal squamous cellcarcinoma; metastatic anal canal carcinoma; recurrent anal canalcarcinoma, recurrent head and neck cancer; head and neck squamous cellcarcinoma (HNSCC), ovarian carcinoma, colon cancer, gastric cancer,advanced GI cancer, gastric adenocarcinoma; gastroesophageal junctionadenocarcinoma, bone neoplasms, soft tissue sarcoma; bone sarcoma,thymic carcinoma, urothelial carcinoma, recurrent merkel cell carcinoma;stage III merkel cell carcinoma; stage IV merkel cell carcinoma,myelodysplastic syndrome and recurrent mycosis fungoides and Sezarysyndrome.

In a related aspect, the disclosure provides a method for treatingcancer or preventing the reoccurrence of cancer comprising administeringto a subject in need thereof a therapeutically effective amount of theantibodies or pharmaceutical compositions contemplated herein. Incertain embodiments, the cancer is selected from the group consisting ofnon small cell lung carcinoma (NSCLC), head and neck cancer, skincancer, melanoma and squamous cell carcinoma (SCC).

In certain embodiments, the cancer has a mutation in the V-Ki-ras2Kirsten rat sarcoma viral oncogene homolog (KRAS) oncogene.

In certain embodiments, the cancer has a mutation in the Harvey ratsarcoma viral oncogene homolog (HRAS) oncogene.

In certain embodiments, the cancer has a mutation in the neuroblastomaRAS viral (v-ras) oncogene homolog (NRAS) oncogene.

In certain embodiments, the cancer has mutations in the RAS oncogene.

In a related aspect, the cancer is selected from the group consisting oflung adenocarcinoma, mucinous adenoma, ductal carcinoma of the pancreasand colorectal carcinoma, brain lower grade glioma, breast invasivecarcinoma, glioblastoma multiforme, melanoma, thyroid, rectumadenocarcinoma, kidney cancer, renal cancer, liver cancer, acute myeloidleukemia, gastric adenocarcinoma, esophageal adenocarcinoma, uterinecorpus endometrioid carcinoma, bladder cancer, prostate cancer, oralcancer, large intestine cancer and lymphoma.

It is contemplated that the methods herein reduce tumor size or tumorburden in the subject, and/or reduce metastasis in the subject. Invarious embodiments, the methods reduce the tumor size by 10%, 20%, 30%or more. In various embodiments, the methods reduce tumor size by 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95% or 100%.

It is contemplated that the methods herein reduce tumor burden, and alsoreduce or prevent the recurrence of tumors once the cancer has gone intoremission.

In another aspect, the disclosure provides a method for treating adisease, condition or disorder associated with TGFβ and PD-1 signalingand/or expression comprising the step of administering to a subject inneed thereof a therapeutically effective amount of an antibody or apharmaceutical composition contemplated herein. In certain embodiments,the disease, condition or disorder is selected from a group of cancers.

The disclosure further contemplates a sterile pharmaceutical compositioncomprising a TGFβ inhibitor, a PD-1 inhibitor and a pharmaceuticallyacceptable carrier.

The disclosure further contemplates a sterile pharmaceutical compositioncomprising a separate TGFβ inhibitor and a pharmaceutically acceptablecarrier.

The disclosure further contemplates a sterile pharmaceutical compositioncomprising a separate PD-1 inhibitor and a pharmaceutically acceptablecarrier.

It is contemplated that inhibitors, such as antibodies, of the presentdisclosure may be given simultaneously, in the same formulation. It isfurther contemplated that the inhibitors are administered in a separateformulation and administered concurrently, with concurrently referringto agents given within 30 minutes of each other. It is furthercontemplated that a third agent may be given simultaneously with theinhibitors.

In another aspect, the disclosure provides a method for treating orpreventing recurrence of a disease, condition or disorder associatedwith TGFβ and PD-1 signaling and/or expression comprising the step ofadministering to a subject in need thereof a therapeutically effectiveamount of an antibody or a pharmaceutical composition contemplatedherein wherein the administration prevents the reocurrence of cancer ina subject that has received inhibitor therapy.

In some embodiments, the TGFβ antibody and/or PD-1 antibody andcombinations thereof or compositions described herein increases thenumber of natural killer (NK) cells in a tumor. In various embodiments,the antibody or composition increases cytolytic activity of NK cells.For example, in various embodiments, the antibodies or compositiondescribed herein increases perforin and granzyme production by NK cells.

In various embodiments, the TGFβ antibody and/or PD-1 antibody andcombinations thereof or compositions described herein decreases thenumber of regulatory T cells in a tumor and/or inhibits regulatory Tcell function. For example, in various embodiments, the antibodies orcomposition described herein inhibits the ability of Tregs todown-regulate an immune response or to migrate to a site of an immuneresponse.

In various embodiments, the TGFβ antibody and/or PD-1 antibody andcombinations thereof or compositions described herein increases thenumber of cytotoxic T cells in a tumor and/or enhances CTL activity,e.g., boosts, increases or promotes CTL activity. For example, invarious embodiments, the antibodies or composition described hereinincreases perforin and granzyme production by CTL and increasescytolytic activity of the CTL.

In various embodiments, the TGFβ antibody and/or PD-1 antibody andcombinations thereof or compositions described herein increases thenumber of dendritic cells (DC) in a tumor and/or inhibits thetolerogenic function (e.g., tolerogenic effect) of dendritic cells. Forexample, in various embodiments, the antibodies or composition describedherein decreases the toleragenic effect of CD8+ dendritic cells.

In various embodiments, administration of the TGFβ antibody and/or PD-1antibody and combinations thereof or compositions described hereinincreases the ratio of effector T cells to regulatory T cells in atumor.

In various embodiments, the disclosure provides a method for increasingthe ratio of effector T cells to regulatory T cells in a tumorcomprising administering to a subject in need thereof therapeuticallyeffective amounts of an inhibitor of transforming growth factor beta(TGFβ) and an inhibitor of Programmed cell death protein 1 (PD-1).

In various embodiments, therapy is administered on a period basis, forexample, hourly, daily, twice weekly, weekly, every 2 weeks, every 3weeks, monthly, once every two months or at a longer interval. In arelated embodiment, in exemplary treatments, a TGFβ inhibitor isadministered in a dose range of 0.1 to 15 mg/kg and a PD-1 inhibitor isadministered in a dose range from 0.1 to 15 mg/kg. These concentrationsmay be administered as a single dosage form or as multiple doses.

In various embodiments, the inhibitors are administered with a thirdagent. In one embodiment, the third agent is selected from the groupconsisting of an extracellular matrix degrading protein, ananti-fibrotic agent, surgical therapy, chemotherapy (e.g. cisplatin pluspemetrexed, carboplatin plus paclitaxel), a cytotoxic agent (e.g.lenalidomide, dexamethasone), or radiation therapy (Philips and Atkins,Int Immunol., 27(1):39-46 (2015) which is incorporated herein byreference). Exemplary third agents are disclosed in greater detail inthe Detailed Description.

Also contemplated is a composition comprising any of the foregoingantibodies or compositions of the disclosure that bind TGFβ or PD-1, oruse thereof in preparation of a medicament, for treatment of any of thedisorders described herein associated with TGFβ and PD-1 signalingand/or expression. Syringes, e.g., single use or pre-filled syringes,sterile sealed containers, e.g. vials, bottle, vessel, and/or kits orpackages comprising any of the foregoing antibodies or compositions,optionally with suitable instructions for use, are also contemplated.

It is understood that each feature or embodiment, or combination,described herein is a non-limiting, illustrative example of any of theaspects of the invention and, as such, is meant to be combinable withany other feature or embodiment, or combination, described herein. Forexample, where features are described with language such as “oneembodiment”, “some embodiments”, “certain embodiments”, “furtherembodiment”, “specific exemplary embodiments”, and/or “anotherembodiment”, each of these types of embodiments is a non-limitingexample of a feature that is intended to be combined with any otherfeature, or combination of features, described herein without having tolist every possible combination. Such features or combinations offeatures apply to any of the aspects of the invention. Where examples ofvalues falling within ranges are disclosed, any of these examples arecontemplated as possible endpoints of a range, any and all numericvalues between such endpoints are contemplated, and any and allcombinations of upper and lower endpoints are envisioned.

In one aspect, the TGFβ antibody useful in the methods is selected fromthe group consisting of XPA.42.089, XPA.42.068 and XPA.42.681. Heavy andlight chain amino acid sequences of XPA.42.089 are set out in SEQ IDNOs: 6 and 8, respectively. Heavy and light chain amino acid sequencesof XPA.42.068 are set out in SEQ ID NOs: 2 and 4, respectively, andheavy and light chain amino acid sequences of XPA.42.681 are set out inSEQ ID NOs: 10 and 12, respectively.

In one aspect, the TGFβ antibody useful in the methods is Fresolimumab(GC1008, Cambridge Antibody Technology, Genzyme and Sanofi), currentlyin Phase I clinical trials (Morris J C et al., PLoS One. 2014 Mar.11;9(3):e90353, 2014; Akhurst and Hata, Nat Rev Drug Discov., 11:790-811, 2012) see U.S. Pat. No. 7,723,486.

In one aspect, the PD-1 antibody useful in the methods is selected fromthe group consisting of pembrolizumab, nivolumab and pidilizumab.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-1C show tumor inhibition in a allograft mouse model with TGFβand PD-1 inhibitor mono- and combination therapy. FIG. 1A shows growthof tumors in mice implanted with cSCC cells. Also shown is the amount ofCD45+, Natural killer (NK) cells, regulatory T (Treg) cells, CD4+ andCD8+ T cells represented as a percentage of viable cells (FIG. 1B) andas a percentage of CD45+ cells (FIG. 1C).

FIG. 2A-2C show the differential responses of individual tumors toimmunotherapy. Data from FIG. 1 were segregated into “responders” and“progressors” to demonstrate the range of responses to α-PD-1 (FIG. 2A),α-TGFβ (FIG. 2B), and combination therapy (FIG. 2C).

FIG. 3A-3D show the differential responses of individual tumors toimmunotherapy. Data from FIG. 1 were plotted as separate tumormeasurements over time for control (ctrl) (FIG. 3A), α-PD-1 monotherapy(FIG. 3B), α-TGFβ monotherapy (FIG. 3C), and α-PD-1 and α-TGFβ incombination (FIG. 3D).

FIG. 4 shows tumor immunotyping measurement of CD45+, Natural killer(NK) cells, regulatory T (Treg) cells, CD4+ and CD8+ T cells tumorschemically-induced (DMBA-TPA) cutaneous squamous cell carcinoma (cSCC)mice, represented as a percentage of viable cells and as a percentage ofCD45+ cells.

FIG. 5 shows the number of mutations per megabase (MB) and sensitivityto TGFβ and PD-1 inhibitor monotherapy and combinational therapy forchemically-induced (DMBA/TPA) Kras- and Hras-driven (FVB-62, FVB-85,FVB-166, FVB-168, FVB-169) and genetically-initiated (GEMM) Kras-drivencSCC cell lines (FVB-1425, FVB-1428) in syngeneic FVB/N mice.

FIG. 6A-6C show the response of the chemically-induced SCC tumor cellline, FVB-168, to pan specific α-TGFβ1, 2, 3 and α-PD1 monotherapy andcombinational therapy. FIG. 6A shows the percentage of carcinomasdisplaying disease progression (continued tumor growth), completeresponse (i.e. inhibition/regression of carcinoma growth) or partialresponse to the indicated therapies. FIG. 6B shows the percentagesurvival of mice in response to pan specific α-TGFβ1, 2, 3 and α-PD1monotherapy and combinational therapy in this model. FIG. 6C shows thelevels of immune cell markers, CD8+ effector (Teff), CD4+ effector(Teff), CD4+ regulatory T (Treg) cells as a percentage of CD45+ and theratio of Teff/Treg for tumors responding and not responding (i.e.progressing tumor growth) to treatment with pan specific α-TGFβ1, 2, 3and PD-1 inhibitor monotherapy and combinational therapy.

FIG. 7 shows the tumor inhibition in an allograft mouse model withTGFβ1, 2 specific, pan-specific TGFβ1, 2, 3, and PD-1 inhibitor mono-and combination therapy. Represented as tumor volume (mm3) over 0-25days post the indicated treatments.

DETAILED DESCRIPTION

The present disclosure provides therapeutics for treating cancer orpreventing the recurrence of cancer. The present disclosure providesmolecules or agents that interact with TGFβ and PD-1 and inhibit one ormore of their functional effects, such as for example signaling throughbinding partners of TGFβ or PD-1. The compositions disclosed hereinadvantageously have the ability to modulate immune cell activity intumors, thereby providing, in one aspect, a method to treat cancer byaffecting a cell population that directly or indirectly affects growthof the tumor.

Definitions

As used herein “TGFβ” refers to any one or more isoforms of TGFβ,including TGFβ1, TGFβ2 and TGFβ3 or variants thereof. Likewise, the term“TGFβ receptor,” unless otherwise indicated, refers to any receptor thatbinds at least one TGFβ isoform.

As used herein “Programmed cell death protein 1” or “PD-1” refers to acell surface receptor involved in immune checkpoint blockade mediated bybinding to two ligands, PD-L1 and PD-L2. PD-1 binding to its ligands hasbeen shown to reduce T-cell proliferation, cytokine production, andcytotoxic activity.

As used herein, the “desired biological activity” of an anti-targetantibody is the ability to bind to TGFβ or PD-1 and inhibit one or moreof their functional effects.

As used herein, a “condition” or “disorder” associated with a “target”in which modification of target activity by a target inhibitor describedherein is beneficial and also includes other disorders in which highlevels of target have been shown to be or are suspected of being eitherresponsible for the pathophysiology of the disorder or a factor thatcontributes to a worsening of the disorder, as well as diseases andother disorders in which modulation of the target is associated withchanges in clinical signs or symptoms. Such disorders may be evidenced,for example, by an increase in the levels of target secreted and/or onthe cell surface and/or modified target signaling in the affected cellsor tissues of a subject suffering from the disorder.

Exemplary diseases, conditions or disorders that can be treated with aninhibitor that inhibits TGFβ and an inhibitor that inhibits PD-1 or aninhibitor of both TGFβ and PD-1 (e.g., antibodies described herein)include cancers, such as esophageal cancer, pancreatic cancer,metastatic pancreatic cancer, metastatic adenocarcinoma of the pancreas,bladder cancer, stomach cancer, fibrotic cancer, glioma, malignantglioma, diffuse intrinsic pontine glioma, recurrent childhood brainneoplasm renal cell carcinoma, clear-cell metastatic renal cellcarcinoma, kidney cancer, prostate cancer, metastatic castrationresistant prostate cancer, stage IV prostate cancer, metastaticmelanoma, melanoma, malignant melanoma, recurrent melanoma of the skin,melanoma brain metastases, stage IIIA skin melanoma; stage IIIB skinmelanoma, stage IIIC skin melanoma; stage IV skin melanoma, malignantmelanoma of head and neck, lung cancer, non small cell lung cancer(NSCLC), squamous cell non-small cell lung cancer, breast cancer,recurrent metastatic breast cancer, hepatocellular carcinoma, hodgkin'slymphoma, follicular lymphoma, non-hodgkin's lymphoma, advanced B-cellNHL, HL including diffuse large B-cell lymphoma (DLBCL), multiplemyeloma, chronic myeloid leukemia, adult acute myeloid leukemia inremission; adult acute myeloid leukemia with Inv(16)(p13.1q22);CBFB-MYH11; adult acute myeloid leukemia with t(16;16)(p13.1;q22);CBFB-MYH11; adult acute myeloid leukemia with t(8;21)(q22;q22);RUNX1-RUNX1T1; adult acute myeloid leukemia with t(9;11)(p22;q23);MLLT3-MLL; adult acute promyelocytic leukemia with t(15;17)(q22;q12);PML-RARA; alkylating agent-related acute myeloid leukemia, chroniclymphocytic leukemia, richter's syndrome; waldenstrom macroglobulinemia,adult glioblastoma; adult gliosarcoma, recurrent glioblastoma, recurrentchildhood rhabdomyosarcoma, recurrent ewing sarcoma/peripheral primitiveneuroectodermal tumor, recurrent neuroblastoma; recurrent osteosarcoma,colorectal cancer, MSI positive colorectal cancer; MSI negativecolorectal cancer, nasopharyngeal nonkeratinizing carcinoma; recurrentnasopharyngeal undifferentiated carcinoma, cervical adenocarcinoma;cervical adenosquamous carcinoma; cervical squamous cell carcinoma;recurrent cervical carcinoma; stage IVA cervical cancer; stage IVBcervical cancer, anal canal squamous cell carcinoma; metastatic analcanal carcinoma; recurrent anal canal carcinoma, recurrent head and neckcancer; carcinoma, squamous cell of head and neck, head and necksquamous cell carcinoma (HNSCC), ovarian carcinoma, colon cancer,gastric cancer, advanced GI cancer, gastric adenocarcinoma;gastroesophageal junction adenocarcinoma, bone neoplasms, soft tissuesarcoma; bone sarcoma, thymic carcinoma, urothelial carcinoma, recurrentmerkel cell carcinoma; stage III merkel cell carcinoma; stage IV merkelcell carcinoma, myelodysplastic syndrome and recurrent mycosis fungoidesand Sezary syndrome.

An “immunoglobulin” or “native antibody” is a tetrameric glycoprotein.In a naturally-occurring immunoglobulin, each tetramer is composed oftwo identical pairs of polypeptide chains, each pair having one “light”(about 25 kDa) and one “heavy” chain (about 50-70 kDa). Theamino-terminal portion of each chain includes a variable region of about100 to 110 or more amino acids primarily responsible for antigenrecognition. The carboxy-terminal portion of each chain defines aconstant region primarily responsible for effector function. Human lightchains are classified as kappa (κ) and lambda (λ) light chains. Heavychains are classified as mu (μ), delta (Δ), gamma (γ), alpha (α), andepsilon (ε), and define the antibody's isotype as IgM, IgD, IgG, IgA,and IgE, respectively. Within light and heavy chains, the variable andconstant regions are joined by a “J” region of about 12 or more aminoacids, with the heavy chain also including a “D” region of about 10 moreamino acids. See generally, Fundamental Immunology, Ch. 7 (Paul, W.,ed., 2nd ed. Raven Press, N.Y. (1989)) (incorporated by reference in itsentirety for all purposes). The variable regions of each light/heavychain pair form the antibody binding site such that an intactimmunoglobulin has two binding sites.

Each heavy chain has at one end a variable domain (VH) followed by anumber of constant domains. Each light chain has a variable domain atone end (VL) and a constant domain at its other end; the constant domainof the light chain is aligned with the first constant domain of theheavy chain, and the light chain variable domain is aligned with thevariable domain of the heavy chain. Particular amino acid residues arebelieved to form an interface between the light and heavy chain variabledomains (Chothia et al., J. Mol. Biol. 196:901-917, 1987).

Immunoglobulin variable domains exhibit the same general structure ofrelatively conserved framework regions (FR) joined by threehypervariable regions or CDRs. From N-terminus to C-terminus, both lightand heavy chains comprise the domains FR1, CDR1, FR2, CDR2, FR3, CDR3and FR4. The assignment of amino acids to each domain is in accordancewith the definitions of Kabat Sequences of Proteins of ImmunologicalInterest (National Institutes of Health, Bethesda, Md. (1987 and 1991)),or Chothia & Lesk, (J. Mol. Biol. 196:901-917, 1987); Chothia et al.,(Nature 342:878-883, 1989).

The hypervariable region of an antibody refers to the CDR amino acidresidues of an antibody which are responsible for antigen-binding. Thehypervariable region comprises amino acid residues from a CDR [e.g.,residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chainvariable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the heavychain variable domain as described by Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)] and/or thoseresidues from a hypervariable loop (e.g., residues 26-32 (L1), 50-52(L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1),53-55 (H2) and 96-101 (H3) in the heavy chain variable domain asdescribed by [Chothia et al., J. Mol. Biol. 196: 901-917 (1987)]. CDRshave also been identified and numbered according to ImMunoGenTics (IMGT)numbering (Lefranc, M.-P., The Immunologist, 7, 132-136 (1999); Lefranc,M.-P. et al., Dev. Comp. Immunol., 27, 55-77 (2003), which describes theCDR locations in the light and heavy chain variable domains as follows:CDR1, approximately residues 27 to 38; CDR2, approximately residues 56to 65; and, CDR3, approximately residues 105 to 116 (germline) orresidues 105 to 117 (rearranged). In one embodiment, it is contemplatedthat the CDRs are located at approximately residues 26-31 (L1), 49-51(L2) and 88-98 (L3) in the light chain variable domain and approximatelyresidues 26-33 (H1), 50-58 (H2) and 97-111 (H3) in the heavy chainvariable domain of an antibody heavy or light chain of approximatelysimilar length to those disclosed herein. However, one of skill in theart understands that the actual location of the CDR residues may varyfrom the projected residues described above when the sequence of theparticular antibody is identified.

Framework or FR residues are those variable domain residues other thanthe hypervariable region residues.

“Heavy chain variable region” as used herein refers to the region of theantibody molecule comprising at least one complementarity determiningregion (CDR) of said antibody heavy chain variable domain. The heavychain variable region may contain one, two, or three CDR of saidantibody heavy chain.

“Light chain variable region” as used herein refers to the region of anantibody molecule, comprising at least one complementarity determiningregion (CDR) of said antibody light chain variable domain. The lightchain variable region may contain one, two, or three CDRs of saidantibody light chain, which may be either a kappa or lambda light chaindepending on the antibody.

The term “antibody” is used in the broadest sense and includes fullyassembled antibodies, tetrameric antibodies, monoclonal antibodies,polyclonal antibodies, multispecific antibodies (e.g., bispecificantibodies), antibody fragments that can bind an antigen (e.g., Fab′,F′(ab)2, Fv, single chain antibodies, diabodies), and recombinantpeptides comprising the forgoing as long as they exhibit the desiredbiological activity. An “immunoglobulin” or “tetrameric antibody” is atetrameric glycoprotein that consists of two heavy chains and two lightchains, each comprising a variable region and a constant region.Antigen-binding portions may be produced by recombinant DNA techniquesor by enzymatic or chemical cleavage of intact antibodies. Antibodyfragments or antigen-binding portions include, inter alia, Fab, Fab′,F(ab′)2, Fv, domain antibody (dAb), complementarity determining region(CDR) fragments, CDR-grafted antibodies, single-chain antibodies (scFv),single chain antibody fragments, chimeric antibodies, diabodies,triabodies, tetrabodies, minibody, linear antibody; chelatingrecombinant antibody, a tribody or bibody, an intrabody, a nanobody, asmall modular immunopharmaceutical (SMIP), an antigen-binding-domainimmunoglobulin fusion protein, a camelized antibody, a VHH containingantibody, or a variant or a derivative thereof, and polypeptides thatcontain at least a portion of an immunoglobulin that is sufficient toconfer specific antigen binding to the polypeptide, such as one, two,three, four, five or six CDR sequences, as long as the antibody retainsthe desired biological activity.

“Monoclonal antibody” refers to an antibody obtained from a populationof substantially homogeneous antibodies, i.e., the individual antibodiescomprising the population are identical except for possible naturallyoccurring mutations that may be present in minor amounts.

“Antibody variant” as used herein refers to an antibody polypeptidesequence that contains at least one amino acid substitution, deletion,or insertion in the variable region of the reference antibody variableregion domains. Variants may be substantially homologous orsubstantially identical to the unmodified antibody.

A “chimeric antibody,” as used herein, refers to an antibody containingsequence derived from two different antibodies (see, e.g., U.S. Pat. No.4,816,567) which typically originate from different species. Mosttypically, chimeric antibodies comprise human and rodent antibodyfragments, generally human constant and mouse variable regions.

A “neutralizing antibody” is an antibody molecule which is able toeliminate or significantly reduce a biological function of a targetantigen to which it binds. Accordingly, a “neutralizing” anti-targetantibody is capable of eliminating or significantly reducing abiological function, such as enzyme activity, ligand binding, orintracellular signaling.

An “isolated” antibody is one that has been identified and separated andrecovered from a component of its natural environment. Contaminantcomponents of its natural environment are materials that would interferewith diagnostic or therapeutic uses for the antibody, and may includeenzymes, hormones, and other proteinaceous or non-proteinaceous solutes.In preferred embodiments, the antibody will be purified (1) to greaterthan 95% by weight of antibody as determined by the Lowry method, andmost preferably more than 99% by weight, (2) to a degree sufficient toobtain at least 15 residues of N-terminal or internal amino acidsequence by use of a spinning cup sequenator, or (3) to homogeneity bySDS-PAGE under reducing or nonreducing conditions using Coomassie blueor, preferably, silver stain. Isolated antibody includes the antibody insitu within recombinant cells since at least one component of theantibody's natural environment will not be present. Ordinarily, however,isolated antibody will be prepared by at least one purification step.

As used herein, an antibody that “specifically binds” is “targetspecific”, is “specific for” target or is “immunoreactive” with thetarget antigen refers to an antibody or antibody substance that bindsthe target antigen with greater affinity than with similar antigens. Inone aspect of the disclosure, the target-binding polypeptides, orfragments, variants, or derivatives thereof, will bind with a greateraffinity to human target as compared to its binding affinity to targetof other, i.e., non-human, species, but binding polypeptides thatrecognize and bind orthologs of the target are within the scopeprovided.

For example, a polypeptide that is an antibody or fragment thereof“specific for” its cognate antigen indicates that the variable regionsof the antibodies recognize and bind the polypeptide of interest with adetectable preference (i.e., able to distinguish the polypeptide ofinterest from other known polypeptides of the same family, by virtue ofmeasurable differences in binding affinity, despite the possibleexistence of localized sequence identity, homology, or similaritybetween family members). It will be understood that specific antibodiesmay also interact with other proteins (for example, S. aureus protein Aor other antibodies in ELISA techniques) through interactions withsequences outside the variable region of the antibodies, and inparticular, in the constant region of the molecule. Screening assays todetermine binding specificity of an antibody for use in the methods ofthe present disclosure are well known and routinely practiced in theart. For a comprehensive discussion of such assays, see Harlow et al.(Eds), Antibodies A Laboratory Manual; Cold Spring Harbor Laboratory;Cold Spring Harbor, N.Y. (1988), Chapter 6. Antibodies for use in themethods can be produced using any method known in the art.

The term “epitope” refers to that portion of any molecule capable ofbeing recognized by and bound by a selective binding agent at one ormore of the antigen binding regions. Epitopes usually consist ofchemically active surface groupings of molecules, such as, amino acidsor carbohydrate side chains, and have specific three-dimensionalstructural characteristics as well as specific charge characteristics.Epitopes as used herein may be contiguous or non-contiguous. Moreover,epitopes may be mimetic (mimotopes) in that they comprise a threedimensional structure that is identical to the epitope used to generatethe antibody, yet comprise none or only some of the amino acid residuesfound in the target that were used to stimulate the antibody immuneresponse. As used herein, a mimotope is not considered a differentantigen from the epitope bound by the selective binding agent; theselective binding agent recognizes the same three-dimensional structureof the epitope and mimotope.

The term “derivative” when used in connection with antibody substancesand polypeptides of the present disclosure refers to polypeptideschemically modified by such techniques as ubiquitination, conjugation totherapeutic or diagnostic agents, labeling (e.g., with radionuclides orvarious enzymes), covalent polymer attachment such as pegylation(derivatization with polyethylene glycol) and insertion or substitutionby chemical synthesis of amino acids such as ornithine, which do notnormally occur in human proteins. Derivatives retain the bindingproperties of underivatized molecules of the disclosure.

“Detectable moiety” or a “label” refers to a composition detectable byspectroscopic, photochemical, biochemical, immunochemical, or chemicalmeans. For example, useful labels include 32P, 35S, fluorescent dyes,electron-dense reagents, enzymes (e.g., as commonly used in an ELISA),biotin-streptavadin, dioxigenin, haptens and proteins for which antiseraor monoclonal antibodies are available, or nucleic acid molecules with asequence complementary to a target. The detectable moiety oftengenerates a measurable signal, such as a radioactive, chromogenic, orfluorescent signal, that can be used to quantitate the amount of bounddetectable moiety in a sample.

The term “therapeutically effective amount” is used herein to indicatethe amount of target-specific composition of the disclosure that iseffective to ameliorate or lessen symptoms or signs of disease to betreated.

The terms “treat”, “treated”, “treating” and “treatment”, as used withrespect to methods herein refer to eliminating, reducing, suppressing orameliorating, either temporarily or permanently, either partially orcompletely, a clinical symptom, manifestation or progression of anevent, disease or condition. Such treating need not be absolute to beuseful.

The present methods provides for use of target-specific antibodies,which may comprise those exemplary sequences set out herein, fragments,variants and derivatives thereof, pharmaceutical formulations includinga target-specific antibodies recited herein. Depending on the amino acidsequence of the constant domain of their heavy chains, immunoglobulinscan be assigned to different classes, IgA, IgD, IgE, IgG and IgM, whichmay be further divided into subclasses or isotypes, e.g. IgG1, IgG2,IgG3, IgG4, IgA1 and IgA2. The subunit structures and three-dimensionalconfigurations of different classes of immunoglobulins are well known.Different isotypes have different effector functions; for example, IgG1and IgG3 isotypes have ADCC activity. An antibody disclosed herein, ifit comprises a constant domain, may be of any of these subclasses orisotypes.

The antibodies used in the present methods may exhibit binding affinityto one or more TGFβ and/or PD-1 antigens of a Kd of less than or equalto about 10⁻⁵ M, less than or equal to about 10⁻⁶ M, or less than orequal to about 10⁻⁷ M, or less than or equal to about 10⁻⁸ M, or lessthan or equal to about 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, or 10⁻¹² M or less.Such affinities may be readily determined using conventional techniques,such as by equilibrium dialysis; by using surface plasmon resonance(SPR) technology (e.g., the BIAcore 2000 instrument, using generalprocedures outlined by the manufacturer); by radioimmunoassay using ¹²⁵Ilabeled target antigen; or by another method set forth in the examplesbelow or known to the skilled artisan. The affinity data may beanalyzed, for example, by the method of Scatchard et al., (Ann N.Y.Acad. Sci., 51:660, 1949).

A KinExA kinetic exclusion assay is also useful to measure the affinityof an antibody for its antigen. KinExA technology measures bindingevents in the solution phase, rather than binding events between asolution phase and a solid phase. In addition, while many methods formeasuring binding events require at least one reactant be modifiedthrough immobilization or labeling, the KinExA method does not requiremodification of molecules under study. The KinExA method is believed toallow a wider range of binding constants to be measured than othermethods currently available. Additional description about KinExA devicesand operation for antibody characterization is available from themanufacturer (Sapidyne Instruments, Inc., Boise, Id.) and can be foundin the published literature, for example U.S. Pat. No. 6,664,114 andDarling et al., “Kinetic Exclusion Assay Technology: Characterization ofMolecular Interactions.” Assay and Drug Development Technologies, 2004,2:647-657.

Transforming Growth Factor β

TGFβ is a disulfide linked dimer that is synthesized as a preproproteinof about 400 amino acids (aa) which is cleaved prior to secretion toproduce mature TGFβ. The N-terminal cleavage fragment, known as the“latency-associated peptide” (LAP), may remain noncovalently bound tothe dimer, thereby inactivating TGFβ. TGFβ isolated in vivo, is foundpredominantly in the inactive, “latent” form, i.e., associated with LAP.Latent TGFβ complex may be activated in several ways, for example, bybinding to a cell surface receptor called the cation-independentmannose-6-phosphate/insulin-like growth factor II receptor. Bindingoccurs through mannose-6-phosphate residues attached at glycosylationsites within LAP. Upon binding to the receptor, TGFβ is released in itsmature form. Mature, active TGFβ is then free to bind to its receptorand exert its biological functions. The major TGFβ binding domain in thetype II TGFβ receptor has been mapped to a 19 amino acid sequence(Demetriou et al., J. Biol. Chem., 271:12755, 1996). See also U.S. Pat.Nos. 7,867,496 and 8,569,462.

Currently, there are five known isoforms of TGFβ (TGFβ1 to TGFβ5;TGFβ1-3 are mammalian, TGFβ4 is found in chicken; and TGFβ5 found infrog), all of which are homologous among each other (60-80% identity),form homodimers of about 25 kDa, and act upon common TGFβ receptors(TGFβ-RI, TGFβ-RII, TGFβ-RIIB, and TGFβ-RIII). The structural andfunctional aspects of TGFβ as well as TGFβ receptors are well-known inthe art (see, for example, Cytokine Reference, eds. Oppenheim et al.,Academic Press, San Diego, Calif., 2001). TGFβ is well-conserved amongspecies. For example, the amino acid sequences of rat and human matureTGFβ1s are nearly identical. See also U.S. Pat. No. 7,867,496.

TGFβ1 plays an important role in the process of wound healing inbiological tissues (New Engl. J. Med., Vol. 331, p. 1286, 1994 and J.Cell. Biol., Vol. 119, p. 1017, 1992). At the site of wounded tissue,biological reactions such as infiltration of inflammatory cells andfibroblast cells, production of extracellular rmatrix (ECM) andvascularization, and cell growth for the subsequent tissue regenerationoccur to repair the injured tissue. See also U.S. Pat. No. 7,579,186.

TGFβ2 deficient mice demonstrate significant developmental defects,including heart, lung, craniofacial, limb, spine, eye, ear andurogenital defects (Dunker et al., Eur J Biol 267:6982-8, 2001). TGFβ3deficient mice demonstrate almost 100% lethality by 24 hrs after birth.These mice show significant palate impairment and delayed pulmonarydevelopment (Dunker et al., supra). TGFβ2 has also been implicated inthe development of glaucoma (Luthen-Driscoll, Experimental Eye Res81:1-4, 2005), fibrosis associated with Crohn's Disease (Van Assche etal., Inflamm Bowel Dis. 10:55-60, 2004), in wound healing and diabeticnephropathy (Pohlers et al., Biochim Biophys Acta 1792:746-56, 2009)

It has been observed that many human tumors (deMartin et al., EMBO J.,6: 3673 (1987), Kuppner et al., Int. J. Cancer, 42: 562 (1988)) and manytumor cell lines (Derynck et al., Cancer Res., 47: 707 (1987), Robertset al., Br. J. Cancer, 57: 594 (1988)) produce TGFβ.

TGFβ isoform expression in cancer is complex and variable with differentcombinations of TGFβ isoforms having different roles in particularcancers. See e.g., U.S. Pat. No. 7,927,593. For example, TGFβ1 and TGFβ3may play a greater role in ovarian cancer and its progression than TGβ2;while TGFβ1 and TGFβ2 expression is greater in higher gradechondrosarcoma tumors than TGβ3. In human breast cancer, TGFβ1 and TGFβ3are highly expressed, with TGFβ3 expression appearing to correlate withoverall survival—patients with node metastasis and positive TGFβ3expression have poor prognostic outcomes. However, in colon cancer,TGFβ1 and TGFβ2 are more highly expressed than TGFβ3 and are present atgreater circulating levels than in cancer-free individuals. In gliomas,TGFβ2 is important for cell migration.

TGFβ Antibodies

The present disclosure encompasses use of amino acid molecules encodingtarget specific antibodies. In exemplary embodiments, a target specificantibody useful in the methods of the disclosure can comprise a humankappa (κ) or a human lambda (λ) light chain or an amino acid sequencederived therefrom, or a human heavy chain or a sequence derivedtherefrom, or both heavy and light chains together in a single chain,dimeric, tetrameric or other form. In some embodiments, a heavy chainand a light chain of a target specific immunoglobulin are differentamino acid molecules. In other embodiments, the same amino acid moleculecontains a heavy chain variable region and a light chain variable regionof a target specific antibody.

In some embodiments, the amino acid sequence of the human anti-targetantibody for TGFβ useful in the methods comprises one or more CDRs ofthe amino acid sequence of the mature (i.e., missing signal sequence)light chain variable region (VL) of antibodies XPA.42.068, XPA.42.089and XPA.42.681 (SEQ ID NOs: 4, 8 and 12 respectively) or variantsthereof, including CDR grafted, modified, humanized, chimeric, or HumanEngineered antibodies or any other variants described herein. In someembodiments, the VL comprises the amino acid sequence from the beginningof the CDR1 to the end of the CDR3 of the light chain of any one of theforegoing antibodies.

In one embodiment, the target specific antibody comprises a light chainCDR1, CDR2 or CDR3 (LCDR1, LCDR2, LCDR3), each of which areindependently selected from the CDR1, CDR2 and CDR3 regions of anantibody having a light chain variable region comprising the amino acidsequence of the VL region set out in SEQ ID NOs: 4, 8 and 12, a nucleicacid encoding the VH region set out in SEQ ID NOs: 4, 8, and 12, orencoded by a nucleic acid molecule encoding the VL region set out in SEQID NOs: 3, 7, and 11. In one embodiment, the light chain CDR1 is fromapproximately residues 24-34, CDR2 is from approximately residues 50-56and CDR3 extends from approximately residues 89-97, according to Chothianumbering. In an alternate embodiment, it is contemplated that the heavychain CDRs are located at approximately residues 27 to 38 (CDR1);approximately residues 56 to 65 (CDR2); and, approximately residues 105to 116 (germline) or residues 105 to 117 (CDR3) according toImMunoGenTics (IMGT) numbering. In one embodiment, it is contemplatedthat the light chain CDRs are located at approximately residues 26-31(L1), 49-51 (L2) and 88-97 (L3) in the light chain variable domain of anantibody light chain of approximately similar length to those disclosedherein. A polypeptide of the target specific antibody may comprise theCDR1, CDR2 and CDR3 regions of an antibody comprising the amino acidsequence of the VL region selected from the group consisting ofXPA.42.068, XPA.42.089 and XPA.42.681.

In some embodiments, the human target specific antibody for TGFβcomprises one or more CDRs of the amino acid sequence of the mature(i.e., missing signal sequence) heavy chain variable region (VH) ofantibody XPA.42.068, XPA.42.089 and XPA.42.681 set out in SEQ ID NOs: 2,6 and 10, respectively, or variants thereof. In some embodiments, the VHcomprises the amino acid sequence from the beginning of the CDR1 to theend of the CDR3 of any one of the heavy chain of the foregoingantibodies.

In one embodiment, the target specific antibody comprises a heavy chainCDR1, CDR2 or CDR3 (HCDR1, HCDR2, HCDR3), each of which areindependently selected from the CDR1, CDR2 and CDR3 regions of anantibody having a heavy chain variable region comprising the amino acidsequence of the VH region set out in SEQ ID NOs: 2, 6, and 10, a nucleicacid encoding the VH region set out in SEQ ID NOs: 2, 6, and 10, orencoded by a nucleic acid molecule encoding the VH region set out in SEQID NOs: 1, 5, and 9. It is further contemplated that a target specificantibody comprises a heavy chain CDR1, CDR2 or CDR3, each of which areindependently selected from the CDR1, CDR2 and CDR3 regions of anantibody having a heavy chain variable region comprising the amino acidsequence of the VH region set out in SEQ ID NOs: 2, 6, and 10. In oneembodiment, the heavy chain CDRs are located according to Chothianumbering: CDR1 is from approximately residues 26-35, CDR2 is fromapproximately residues 50-58 and CDR3 extends from approximatelyresidues 95-102 (or 95-111 or 95-118). In an alternate embodiment, it iscontemplated that the heavy chain CDRs are located at CDR1,approximately residues 27 to 38 (CDR1); approximately residues 56 to 65(CDR2); and, CDR3, approximately residues 105 to 116 (germline) orresidues 105 to 117 CDR3) according to ImMunoGenTics (IMGT) numbering.In one embodiment, it is contemplated that the heavy chain CDRs arelocated at approximately residues 26-33 (H1), 50-58 (H2) and 97-111 (H3)in the heavy chain variable domain of an antibody heavy chain ofapproximately similar length to those disclosed herein. A polypeptide ofthe target specific antibody may comprise the CDR1, CDR2 and CDR3regions of an antibody comprising the amino acid sequence of the VHregion selected from the group consisting of XPA.42.068, XPA.42.089 andXPA.42.681.

In another embodiment, the TGFβ antibody comprises a mature light chainvariable region as disclosed above and a mature heavy chain variableregion as disclosed above, optionally paired with the correspondinglynamed heavy or light chain, or optionally with a different heavy orlight chain.

In exemplary embodiments, the disclosure contemplates use of: amonoclonal antibody that retains any one, two, three, four, five, or sixof HCDR1, HCDR2, HCDR3, LCDR1, LCDR2, or LCDR3 of any one of SEQ ID NOs:13, 19 and 25; 14, 20 and 26; 15, 21 and 27 and SEQ ID NOs: 16, 22 and28; 17, 23 and 29; and 18, 24 and 30, respectively, optionally includingone or two mutations in any of such CDR(s), e.g., a conservative ornon-conservative substitution, and optionally paired. a monoclonalantibody that retains all of HCDR1, HCDR2, HCDR3, or the heavy chainvariable region of any one of SEQ ID NOs: 13, 19 and 25; 14, 20 and 26;and 15, 21 and 27, optionally including one or two mutations in any ofsuch CDR(s), optionally further comprising any suitable heavy chainconstant region, e.g., IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, or IgE,a human sequence thereof, or a hybrid thereof; a monoclonal antibodythat retains all of LCDR1, LCDR2, LCDR3, or the light chain variableregion of any one SEQ ID NOs: 16, 22 and 28; 17, 23 and 29; and 18, 24and 30, optionally including one or two mutations in any of such CDR(s),optionally further comprising any suitable light chain constant region,e.g., a kappa or lambda light chain constant region, a human sequencethereof, or a hybrid thereof.

In some embodiments, an antibody useful in the methods comprises allthree light chain CDRs, all three heavy chain CDRs, or all six CDRs ofthe light and heavy chain. In some exemplary embodiments, two lightchain CDRs from an antibody may be combined with a third light chain CDRfrom a different antibody. Alternatively, a LCDR1 from one antibody canbe combined with a LCDR2 from a different antibody and a LCDR3 from yetanother antibody, particularly where the CDRs are highly homologous.Similarly, two heavy chain CDRs from an antibody may be combined with athird heavy chain CDR from a different antibody; or a HCDR1 from oneantibody can be combined with a HCDR2 from a different antibody and aHCDR3 from yet another antibody, particularly where the CDRs are highlyhomologous.

In some embodiments, an antibody useful in the methods comprises apolypeptide having an amino acid sequence at least about 65%, 70%, 75%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more identical to the heavy chainvariable region set out in SEQ ID NOs: 2, 6, and 10 and/or an amino acidsequence an amino acid sequence at least about 65%, 70%, 75%, 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% or more identical to the light chain variable regionset out in SEQ ID NOs: 4, 8 and 12, the antibody further comprising atleast one, two, three, four, five or all of HCDR1, HCDR2, HCDR3, LCDR1,LCDR2 or LCDR3. In some embodiments, the amino acid sequence withpercentage identity to the light chain variable region may comprise one,two or three of the light chain CDRs. In other embodiments, the aminoacid sequence with percentage identity to the heavy chain variableregion may comprise one, two, or three of the heavy chain CDRs.

In another embodiment, an antibody useful in the methods comprises apolypeptide having an amino acid sequence at least about 65%, 70%, 75%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more identical to all three HCDRs in theheavy chain variable region of an antibody sequence described herein,the CDRs set out in SEQ ID NOs: 13, 19 and 25; 14, 20 and 26; and 15, 21and 27.

In a related embodiment, an antibody useful in the methods comprises apolypeptide having an amino acid sequence at least about 65%, 70%, 75%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more identical to the all three LCDRs inthe light chain variable region of an antibody sequence describedherein, the CDRs set out in SEQ ID NOs: 16, 22 and 28; 17, 23 and 29;and 18, 24 and 30.

In a further embodiment, an antibody useful in the methods comprises apolypeptide having an amino acid sequence at least about 65%, 70%, 75%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or more identical to the all six CDRs inthe heavy chain and light chain variable regions of an antibody sequencedescribed herein, the CDRs set out in SEQ ID NOs: 13, 19 and 25; 14, 20and 26; and 15, 21 27; 16, 22 and 28; 17, 23 and 29; and 18, 24 and 30.

It is contemplated that the antibodies described herein may have one, ortwo or more amino acid substitutions in the CDR regions of the antibody,e.g., non-conservative or conservative substitutions.

In a related embodiment, the residues of the framework are altered. Theheavy chain framework regions which can be altered lie within regionsdesignated H-FR1, H-FR2, H-FR3 and H-FR4, which surround the heavy chainCDR residues, and the residues of the light chain framework regionswhich can be altered lie within the regions designated L-FR1, L-FR2,L-FR3 and L-FR4, which surround the light chain CDR residues. An aminoacid within the framework region may be replaced, for example, with anysuitable amino acid identified in a human framework or human consensusframework.

In exemplary embodiments, an anti-TGFβ antibody described hereinspecifically binds at least one isoform of TGFβ selected from the groupconsisting of TGFβ1, TGβ2, and TGβ3. In other embodiments, the anti-TGFβantibody specifically binds: (a) TGFβ1, TGβ2, and TGFβ3 (“pan-reactiveantibody” or “pan-binding antibody”); (b) TGFβ1 and TGβ2; (c) TGFβ1 andTGβ3; and (d) TGFβ2 and TGβ3. In exemplary embodiments, an anti-TGFβantibody described herein binds at least one isoform of TGFβ with anaffinity of 10⁻⁶ M, 10−7 M, 10−8 M, 10⁻⁹ M, 10⁻¹⁰ M, 10⁻¹¹ M, or 10⁻¹²M, or less (lower meaning higher binding affinity), or optionally bindstwo TGFβ isoforms, or all of TGFβ1, 2, or 3 with an affinity of 10⁻⁶ M.10⁻⁷ M, 10⁻⁸ M, 10⁻⁹ M 10⁻¹⁰ M, 10⁻¹¹ M, or 10⁻¹² M or less for one ormore of the isoforms. In other embodiments, an antibody described hereinbinds to TGFβ1 and TGFβ2 with at least 2-50 fold, 10-100 fold, 2-fold,5-fold, 10-fold, 25-fold, 50-fold or 100-fold, or 20-50%, 50-100%, 20%,25%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% higher affinity (e.g.,preferentially binds to TGFβ1 and TGFβ2) compared to binding to TGβ3.Alternatively, an antibody described herein, binds each of TGFβ isoformsTGFβ1, TGFβ2 and TGFβ3 with an affinity within 3-fold, 5-fold or 10-foldof each other.

In some embodiments, antibody neutralization of TGFβ1 and TGFβ2 is atleast 2-50 fold, 10-100 fold, 2-fold, 5-fold, 10-fold, 25-fold, 50-foldor 100-fold, or 20-50%, 50-100%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%,90% or 100% more potent than neutralization of TGFβ3.

Heavy and light chain amino acid sequences of XPA.42.089 are set out inSEQ ID NOs: 6 and 8, respectively. Heavy and light chain amino acidsequences of XPA.42.068 are set out in SEQ ID NOs: 2 and 4,respectively, and heavy and light chain amino acid sequences ofXPA.42.681 are set out in SEQ ID NOs: 10 and 12, respectively.

Antibody Nucleic Acids

The present disclosure also encompasses use of nucleic acid moleculesencoding target specific antibodies described herein and in the SequenceListing, optionally for recombinantly producing an antibody describedherein or for generating antibody variants. In some embodiments,different nucleic acid molecules encode a heavy chain variable regionand a light chain variable region of a target specific antibody. Inother embodiments, the same nucleic acid molecule encodes a heavy chainand a light chain variable regions of a target specific antibody. In oneembodiment, the nucleic acid encodes a target specific antibody of thepresent disclosure, as well as any of the polypeptides encoded by thenucleic acids described herein.

Nucleic acid sequences encoding anti-TGFβ antibodies contemplated foruse in the methods include all nucleic acid sequences, including thesequences in SEQ ID NOs: 1, 3, 5, 7, 9 and 11 and nucleic acid sequencescomprises degenerate codons based on the diversity of the genetic code,encoding an amino acid sequence of the heavy and light chain variableregions of an antibody described herein or any HCDRs or LCDRs describedherein, and as set out in SEQ ID NOs: 2, 4, 6, 8, 10, 12 and 13-30, aswell as nucleic acids that hybridize under highly stringent conditions,such as those described herein, to a nucleic acid sequence encoding anamino acid sequence of the heavy and light chain variable regions of anantibody described herein or any HCDRs or LCDRs described herein, and asset out in SEQ ID NOs: 2, 4, 6, 8, 10, 12 and 13-30.

Preparation of variants and derivatives of antibodies andantigen-binding compounds of the present invention, including affinitymaturation or preparation of variants or derivatives containing aminoacid analogs, is described in further detail herein. Exemplary variantsinclude those containing a conservative or non-conservative substitutionof a corresponding amino acid within the amino acid sequence, or areplacement of an amino acid with a corresponding amino acid of adifferent human antibody sequence. Variants and fragments of an antibodydisclosed herein are made using methods as described herein and known inthe field of recombinant protein production.

Programmed Cell Death Protein 1 (PD-1)

PD-1, also known as cluster of differentiation 279 (CD279) is a membraneprotein of 268 amino acids. PD-1 is a member of the CD28/CTLA-4 familyof T cell regulator proteins (Ishida Y et al., The EMBO J., 11 (11):3887-95, (1992)). PD-1 is a cell surface co-inhibitory receptorexpressed on CD4+ and CD8+ T cells, B cells and macrophages, and is acomponent of immune checkpoint blockade (Shinohara et al., Genomics.,23(3):704, (1994); Francisco et al., Immunol Rev., 236: 219, (2010)).PD-1 limits the activity of T cells upon interaction with its twoligands PD-L1 (also known as B7-H1; CD274) and PD-L2 (B7-DC; CD273)(Freeman G J et al., J. Exp. Med. 192 (7): 1027-34, 2000; Latchman Y etal., Nat. Immunol., 2 (3): 261-8, 2001; Postow et al., J Clin Oncol.,33: 9, 2015). Interaction of PD-1 with PD-L1 and PD-L2, reduces T cellproliferation, cytokine production, and cytotoxic activity (Freeman G Jet al., J Exp Med., 192:1027-34, (2000); Brown J A et al., J Immunol.,170:1257-66, (2003)).

PD-1 Antibodies

Antibodies to PD-1 have been described in U.S. Pat. Nos. 8,735,553;8,617,546; 8,008,449; 8,741,295; 8,552,154; 8,354,509; 8,779,105;7,563,869; 8,287,856; 8,927,697; 8,088,905; 7,595,048; 8,168,179;6,808,710; 7,943,743; 8,246,955; and 8,217,149.

It is contemplated that any known antibody can be used in the presentmethods. In some embodiments, a murine monoclonal anti-target antibodyfor human PD-1 is used in the present methods. For example, InVivo MAbanti h PD-1 (BioXCell, Clone: RMP1-14, Cat. no.: BE0146). Anti-PD-1antibodies have been shown to be effective in human therapy, see e.g.,pembrolizumab (KEYTRUDA®, Merck Sharp & Dohme Corp.) and nivolumab(Opdivo®, Bristol-Myers Squibb) which are anti-PD-1 antibodies approvedfor use in human therapy. Additional PD-1 antibodies are in clinicaldevelopment, e.g. pidilizumab (CT-011) (CureTech Ltd.).

Alternatively, antibodies to PD-1 are made using techniques known in theart and described herein, including phage display technology, hybridomatechnology, transgenic mouse technology and others.

In various embodiments, a bispecific antibody that binds both a TGFβ anda PD-1 protein is useful in the present methods.

Monoclonal Antibodies

Monoclonal antibody refers to an antibody obtained from a population ofsubstantially homogeneous antibodies. Monoclonal antibodies aregenerally highly specific, and may be directed against a singleantigenic site, in contrast to polyclonal antibody preparations thattypically include different antibodies directed against the same ordifferent determinants (epitopes). In addition to their specificity,monoclonal antibodies are advantageous in that they are synthesized bythe homogeneous culture, uncontaminated by other immunoglobulins withdifferent specificities and characteristics.

Monoclonal antibodies may be made by the hybridoma method firstdescribed by Kohler et al. (Nature, 256:495-7, 1975) (Harlow & Lane;Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press:Cold Spring Harbor, N.Y. (1988); Goding, Monoclonal Antibodies:Principles and Practice, pp. 59-103 (Academic Press, 1986), or may bemade by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567).The monoclonal antibodies may also be isolated from phage antibodylibraries using the techniques described in, for example, Clackson etal., (Nature 352:624-628, 1991) and Marks et al., (J. Mol. Biol.222:581-597, 1991). Additional methods for producing monoclonalantibodies are well-known to a person of ordinary skill in the art.

Monoclonal antibodies, such as those produced by the above methods, aresuitably separated from culture medium, ascites fluid, or serum byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydrophobic interaction chromatography(HIC), ion exchange chromatography, hydroxyapatite chromatography, gelelectrophoresis, dialysis, and/or affinity chromatography.

It is further contemplated that antibodies of the present disclosure maybe used as smaller antigen binding fragments of the antibody that arewell-known in the art and described herein.

Antibody Fragments

Antibody fragments comprise a portion of an intact full length antibody,preferably an antigen binding or variable region of the intact antibody.Examples of antibody fragments include Fab, Fab′, F(ab′)2, and Fvfragments; diabodies; linear antibodies; single-chain antibody molecules(e.g., scFv); multispecific antibody fragments such as bispecfic,trispecific, etc. antibodies (e.g., diabodies, triabodies, tetrabodies);minibody; chelating recombinant antibody; tribodies or bibodies;intrabodies; nanobodies; small modular immunopharmaceuticals (SMIP),binding-domain immunoglobulin fusion proteins; camelized antibodies; VHHcontaining antibodies; and other polypeptides formed from antibodyfragments. See for example Holliger & Hudson (Nat. Biotech. 23:1126-36(2005)).

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, monovalent fragments consisting ofthe VL, VH, CL and CH domains each with a single antigen-binding site,and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields a F(ab′)2 fragment, abivalent fragment comprising two Fab fragments linked by a disulfidebridge at the hinge region, that has two “Single-chain Fv” or “scFv”antibody fragments comprise the VH and VL domains of antibody, whereinthese domains are present in a single polypeptide chain. Preferably, theFv polypeptide further comprises a polypeptide linker between the VH andVL domains that enables the Fv to form the desired structure for antigenbinding, resulting in a single-chain antibody (scFv), in which a VL andVH region are paired to form a monovalent molecule via a syntheticlinker that enables them to be made as a single protein chain (Bird etal., Science 242:423-426, 1988, and Huston et al., Proc. Natl. Acad.Sci. USA 85:5879-5883, 1988). For a review of scFv see Pluckthun, in ThePharmacology of Monoclonal Antibodies, vol. 1 13, Rosenburg and Mooreeds., Springer-Verlag, New York, pp. 269-315 (1994). An Fd fragmentconsists of the VH and CH1 domains.

Additional antibody fragments include a domain antibody (dAb) fragment(Ward et al., Nature 341:544-546, 1989) which consists of a VH domain.Diabodies are bivalent antibodies in which VH and VL domains areexpressed on a single polypeptide chain, but using a linker that is tooshort to allow for pairing between the two domains on the same chain,thereby forcing the domains to pair with complementary domains ofanother chain and creating two antigen binding sites (see e.g., EP404,097; WO 93/11161; Holliger et al., Proc. Natl. Acad. Sci. USA90:6444-6448, 1993, and Poljak et al., Structure 2:1121-1123, 1994).Diabodies can be bispecific or monospecific.

Functional heavy-chain antibodies devoid of light chains are naturallyoccurring in nurse sharks (Greenberg et al., Nature 374:168-73, 1995),wobbegong sharks (Nuttall et al., Mol Immunol. 38:313-26, 2001) andCamelidae (Hamers-Casterman et al., Nature 363: 446-8, 1993; Nguyen etal., J. Mol. Biol. 275: 413, 1998), such as camels, dromedaries, alpacasand llamas. The antigen-binding site is reduced to a single domain, theVHH domain, in these animals. These antibodies form antigen-bindingregions using only heavy chain variable region, i.e., these functionalantibodies are homodimers of heavy chains only having the structure H2L2(referred to as “heavy-chain antibodies” or “HCAbs”). Camelid VHHreportedly recombines with IgG2 and IgG3 constant regions that containhinge, CH2, and CH3 domains and lack a CH1 domain (Hamers-Casterman etal., supra). For example, llama IgG1 is a conventional (H2L2) antibodyisotype in which VH recombines with a constant region that containshinge, CH1, CH2 and CH3 domains, whereas the llama IgG2 and IgG3 areheavy chain-only isotypes that lack CH1 domains and that contain nolight chains. Camelid VHH domains have been found to bind to antigenwith high affinity (Desmyter et al., J. Biol. Chem. 276:26285-90, 2001)and possess high stability in solution (Ewert et al., Biochemistry41:3628-36, 2002). Classical VH-only fragments are difficult to producein soluble form, but improvements in solubility and specific binding canbe obtained when framework residues are altered to be more VHH-like.(See, e.g., Reichman, et al., J Immunol Methods 1999, 231:25-38.)Methods for generating antibodies having camelid heavy chains aredescribed in, for example, in U.S. Patent Publication Nos. 20050136049and 20050037421.

The variable domain of an antibody heavy-chain is the smallest fullyfunctional antigen-binding fragment with a molecular mass of only 15kDa, this entity is referred to as a nanobody (Cortez-Retamozo et al.,Cancer Research 64:2853-57, 2004). A nanobody library may be generatedfrom an immunized dromedary as described in Conrath et al., (AntimicrobAgents Chemother 45: 2807-12, 2001) or using recombinant methods asdescribed in Revets et al, Expert Opin. Biol. Ther. 5(1):111-24 (2005).

Production of bispecific Fab-scFv (“bibody”) and trispecificFab-(scFv)(2) (“tribody”) are described in Schoonjans et al. (J Immunol.165:7050-57, 2000) and Willems et al. (J Chromatogr B Analyt TechnolBiomed Life Sci. 786:161-76, 2003). For bibodies or tribodies, a scFvmolecule is fused to one or both of the VL-CL (L) and VH-CH1 (Fd)chains, e.g., to produce a tribody two scFvs are fused to C-term of Fabwhile in a bibody one scFv is fused to C-term of Fab.

A “minibody” consisting of scFv fused to CH3 via a peptide linker(hingeless) or via an IgG hinge has been described in Olafsen, et al.,Protein Eng Des Sel. 17(4):315-23, 2004.

Intrabodies are single chain antibodies which demonstrate intracellularexpression and can manipulate intracellular protein function (Biocca, etal., EMBO J. 9:101-108, 1990; Colby et al., Proc Natl Acad Sci USA.101:17616-21, 2004). Intrabodies, which comprise cell signal sequenceswhich retain the antibody construct in intracellular regions, may beproduced as described in Mhashilkar et al (EMBO J 14:1542-51, 1995) andWheeler et al. (FASEB J. 17:1733-5. 2003). Transbodies arecell-permeable antibodies in which a protein transduction domain (PTD)is fused with single chain variable fragment (scFv) antibodies Heng etal., (Med Hypotheses. 64:1105-8, 2005).

Further contemplated are antibodies that are SMIPs or binding domainimmunoglobulin fusion proteins specific for target protein. Theseconstructs are single-chain polypeptides comprising antigen bindingdomains fused to immunoglobulin domains necessary to carry out antibodyeffector functions. See e.g., WO03/041600, U.S. Patent publication20030133939 and US Patent Publication 20030118592.

One or more CDRs may be incorporated into a molecule either covalentlyor noncovalently to make it an immunoadhesin. An immunoadhesin mayincorporate the CDR(s) as part of a larger polypeptide chain, maycovalently link the CDR(s) to another polypeptide chain, or mayincorporate the CDR(s) noncovalently. The CDRs permit the immunoadhesinto specifically bind to a particular antigen of interest.

Thus, a variety of compositions comprising one, two, and/or three CDRs(e.g., a single CDR alone or in tandem, 2, 3, or other multiple repeatsof the CDRs; or combinations of 2 or 3 CDRs alone or in tandem repeats;optionally, with a spacer amino acid sequence between the CDRs orrepeats) of a heavy chain variable region or a light chain variableregion of an antibody may be generated by techniques known in the art.

Multispecific Antibodies

In some embodiments, it may be desirable to generate multispecific (e.g.bispecific) anti-target antibody having binding specificities for atleast two different epitopes of the same or different molecules.Exemplary bispecific antibodies may bind to two different epitopes ofthe target molecule. Alternatively, a TGFβ-specific antibody arm may becombined with an arm which binds to PD-1. Bispecific antibodies may alsobe used to localize cytotoxic agents to cells which express or take up atarget. These antibodies possess a target-binding arm and an arm whichbinds the cytotoxic agent (e.g., saporin, anti-interferon-60, vincaalkaloid, ricin A chain, methotrexate or radioactive isotope hapten).Bispecific antibodies can be prepared as full length antibodies orantibody fragments (e.g., F(ab′)2 bispecific antibodies). See alsoreviews of the methods and therapeutic benefits of bispecific antibodiesin Spasevska I, BioSciences Master Reviews, 2014; Caravella J andLugovskoy A, Curr Opin Chem Biol., 14(4) 520-528, 2010

According to another approach for making bispecific antibodies, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers which are recovered fromrecombinant cell culture. The preferred interface comprises at least apart of the CH3 domain of an antibody constant domain. In this method,one or more small amino acid side chains from the interface of the firstantibody molecule are replaced with larger side chains (e.g., tyrosineor tryptophan). Compensatory “cavities” of identical or similar size tothe large side chain(s) are created on the interface of the secondantibody molecule by replacing large amino acid side chains with smallerones (e.g., alanine or threonine). This provides a mechanism forincreasing the yield of the heterodimer over other unwanted end-productssuch as homodimers. See WO96/27011.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Heteroconjugateantibodies may be made using any convenient cross-linking methods.Suitable cross-linking agents are well known in the art, and aredisclosed in U.S. Pat. No. 4,676,980, along with a number ofcross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,(Science 229:81-83, 1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)2 fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes. In yet afurther embodiment, Fab′-SH fragments directly recovered from E. colican be chemically coupled in vitro to form bispecific antibodies.(Shalaby et al., J. Exp. Med. 175:217-225 (1992))

Shalaby et al., J. Exp. Med. 175:217-225 (1992) describe the productionof a fully humanized bispecific antibody F(ab′)2 molecule. Each Fab′fragment was separately secreted from E. coli and subjected to directedchemical coupling in vitro to form the bispecfic antibody. Thebispecific antibody thus formed was able to bind to cells overexpressingthe HER2 receptor and normal human T cells, as well as trigger the lyticactivity of human cytotoxic lymphocytes against human breast tumortargets.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. (Kostelny et al., J. Immunol. 148:1547-1553, 1992). Theleucine zipper peptides from the Fos and Jun proteins were linked to theFab′ portions of two different antibodies by gene fusion. The antibodyhomodimers were reduced at the hinge region to form monomers and thenre-oxidized to form the antibody heterodimers. This method can also beutilized for the production of antibody homodimers. The “diabody”technology described by Hollinger et al. (Proc. Natl. Acad. Sci. USA90:6444-48, 1993) has provided an alternative mechanism for makingbispecific antibody fragments.

The fragments comprise a heavy chain variable region (VH) connected to alight-chain variable region (VL) by a linker which is too short to allowpairing between the two domains on the same chain. Accordingly, the VHand VL domains of one fragment are forced to pair with the complementaryVL and VH domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (scFv) dimers has also beenreported. See Gruber et al., J. Immunol. 152: 5368 (1994).

Alternatively, the bispecific antibody may be a “linear antibody”produced as described in Zapata et al. Protein Eng. 8:1057-62 (1995).Linear antibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1)which form a pair of antigen binding regions. Linear antibodies can bebispecific or monospecific.

In a further embodiment, the bispecific antibody may be a chelatingrecombinant antibody (CRAb). A chelating recombinant antibody recognizesadjacent and non-overlapping epitopes of the target antigen, and isflexible enough to bind to both epitopes simultaneously (Neri et al., JMol Biol. 246:367-73, 1995).

Antibodies with more than two valencies are also contemplated. Forexample, trispecific antibodies can be prepared. (Tutt et al., J.Immunol. 147:60, 1991).

Chimeric and Humanized Antibodies

Because chimeric or humanized antibodies are less immunogenic in humansthan the parental non-human (e.g., mouse) monoclonal antibodies, theycan be used for the treatment of humans with far less risk ofanaphylaxis.

Chimeric monoclonal antibodies, in which the variable Ig domains of anon-human (e.g., mouse)monoclonal antibody are fused to human constantIg domains, can be generated using standard procedures known in the art(See Morrison et al., Proc. Natl. Acad. Sci. USA 81, 6841-6855 (1984);and, Boulianne et al, Nature 312, 643-646, (1984)).

Humanized antibodies may be achieved by a variety of methods including,for example: (1) grafting the non-human complementarity determiningregions (CDRs) onto a human framework and constant region (a processreferred to in the art as humanizing through “CDR grafting”), (2)transplanting the entire non-human variable domains, but “cloaking” themwith a human-like surface by replacement of surface residues (a processreferred to in the art as “veneering”), or, alternatively, (3)substituting human amino acids at positions determined to be unlikely toadversely effect either antigen binding or protein folding, but likelyto reduce immunogenicity in a human environment (e.g., HUMANENGINEERING™). In the present disclosure, humanized antibodies willinclude both “humanized,” “veneered” and “HUMAN ENGINEERED™” antibodies.These methods are disclosed in, e.g., Jones et al., Nature 321:522 525(1986); Morrison et al., Proc. Natl. Acad. Sci., U.S.A., 81:6851-6855(1984); Morrison and Oi, Adv. Immunol., 44:65-92 (1988); Verhoeyer etal., Science 239:1534-1536 (1988); Padlan, Molec. Immun. 28:489-498(1991); Padlan, Molec. Immunol. 31:169-217 (1994); Studnicka et al. U.S.Pat. No. 5,766,886; Studnicka et al., (Protein Engineering 7: 805-814,1994; Co et al., J. Immunol. 152, 2968-2976 (1994); Riechmann, et al.,Nature 332:323-27 (1988); and Kettleborough et al., Protein Eng.4:773-783 (1991) each of which is incorporated herein by reference. CDRgrafting techniques are known in the field, see for example, Riechmann,et al. (Nature 332:323-27 (1988)).

Human Antibodies from Transgenic Animals

Human antibodies to target protein can also be produced using transgenicanimals that have no endogenous immunoglobulin production and areengineered to contain human immunoglobulin loci. For example, WO98/24893 discloses transgenic animals having a human Ig locus whereinthe animals do not produce functional endogenous immunoglobulins due tothe inactivation of endogenous heavy and light chain loci. WO 91/00906also discloses transgenic non-primate mammalian hosts capable ofmounting an immune response to an immunogen, wherein the antibodies haveprimate constant and/or variable regions, and wherein the endogenousimmunoglobulin encoding loci are substituted or inactivated. WO 96/30498and U.S. Pat. No. 6,091,001 disclose the use of the Cre/Lox system tomodify the immunoglobulin locus in a mammal, such as to replace all or aportion of the constant or variable region to form a modified antibodymolecule. WO 94/02602 discloses non-human mammalian hosts havinginactivated endogenous Ig loci and functional human Ig loci. U.S. Pat.No. 5,939,598 discloses methods of making transgenic mice in which themice lack endogenous heavy chains, and express an exogenousimmunoglobulin locus comprising one or more xenogeneic constant regions.See also, U.S. Pat. Nos. 6,114,598 6,657,103 and 6,833,268; Green L L,Curr Drug Discovery Technol., 11(1), 74-84, 2014; Lee E C et al., NatureBiotechnology, 32:356-363, 2014; Lee E C and Owen M, Methods Mol Biol.,901:137-48, 2012).

Using a transgenic animal described above, an immune response can beproduced to a selected antigenic molecule, and antibody producing cellscan be removed from the animal and used to produce hybridomas thatsecrete human monoclonal antibodies. Immunization protocols, adjuvants,and the like are known in the art, and are used in immunization of, forexample, a transgenic mouse as described in WO 96/33735. Thispublication discloses monoclonal antibodies against a variety ofantigenic molecules including IL-6, IL-8, TNFa, human CD4, L selectin,gp39, and tetanus toxin. The monoclonal antibodies can be tested for theability to inhibit or neutralize the biological activity orphysiological effect of the corresponding protein. WO 96/33735 disclosesthat monoclonal antibodies against IL-8, derived from immune cells oftransgenic mice immunized with IL-8, blocked IL-8 induced functions ofneutrophils. Human monoclonal antibodies with specificity for theantigen used to immunize transgenic animals are also disclosed in WO96/34096 and U.S. patent application no. 20030194404; and U.S. patentapplication no. 20030031667.

Additional transgenic animals useful to make monoclonal antibodiesinclude the Medarex HuMAb-MOUSE®, described in U.S. Pat. No. 5,770,429and Fishwild, et al. (Nat. Biotechnol. 14:845-851 (1996)), whichcontains gene sequences from unrearranged human antibody genes that codefor the heavy and light chains of human antibodies. Immunization of aHuMAb-MOUSE® enables the production of fully human monoclonal antibodiesto the target protein.

Also, Ishida et al. (Cloning Stem Cells. 4:91-102 (2002)) describes theTransChromo Mouse (TCMOUSE™) which comprises megabase-sized segments ofhuman DNA and which incorporates the entire human immunoglobulin (hlg)loci. The TCMOUSE™ has a fully diverse repertoire of hlgs, including allthe subclasses of IgGs (IgG1-G4). Immunization of the TCMOUSE™ withvarious human antigens produces antibody responses comprising humanantibodies.

See also Jakobovits et al., Proc. Natl. Acad. Sci. USA, 90:2551 (1993);Jakobovits et al., Nature, 362:255-258 (1993); Bruggermann et al., Yearin Immunol., 7:33 (1993); and U.S. Pat. Nos. 5,591,669, 5,589,369,5,545,807; and U.S Patent Publication No. 20020199213. U.S. PatentPublication No. 20030092125 describes methods for biasing the immuneresponse of an animal to the desired epitope. Human antibodies may alsobe generated by in vitro activated B cells (see U.S. Pat. Nos. 5,567,610and 5,229,275).

Human Antibodies from Display Technology

The development of technologies for making repertoires of recombinanthuman antibody genes, and the display of the encoded antibody fragmentson the surface of filamentous bacteriophage, has provided a means formaking human antibodies directly. Antibodies produced by phagetechnology are produced as antigen binding fragments-usually Fv or Fabfragments-in bacteria and thus lack effector functions. Effectorfunctions can be introduced by one of two strategies: The fragments canbe engineered, for example, into complete antibodies for expression inmammalian cells, or into bispecific antibody fragments with a secondbinding site capable of triggering an effector function.

By way of example, one method for preparing the library of antibodiesfor use in phage display techniques comprises the steps of immunizing anon-human animal comprising human immunoglobulin loci with targetantigen or an antigenic portion thereof to create an immune response,extracting antibody producing cells from the immunized animal; isolatingRNA from the extracted cells, reverse transcribing the RNA to producecDNA, amplifying the cDNA using a primer, and inserting the cDNA into aphage display vector such that antibodies are expressed on the phage.Recombinant target-specific antibodies of the disclosure may be obtainedin this way.

In another example, antibody producing cells can be extracted fromnon-immunized animals, RNA isolated from the extracted cells and reversetranscribed to produce cDNA, which is amplified using a primer, andinserted into a phage display vector such that antibodies are expressedon the phage. Phage-display processes mimic immune selection through thedisplay of antibody repertoires on the surface of filamentousbacteriophage, and subsequent selection of phage by their binding to anantigen of choice. One such technique is described in WO 99/10494, whichdescribes the isolation of high affinity and functional agonisticantibodies for MPL and msk receptors using such an approach. Antibodiesof the disclosure can be isolated by screening of a recombinantcombinatorial antibody library, preferably a scFv phage display library,prepared using human VL and VH cDNAs prepared from mRNA derived fromhuman lymphocytes. Methodologies for preparing and screening suchlibraries are known in the art. See e.g., U.S. Pat. No. 5,969,108. Thereare commercially available kits for generating phage display libraries(e.g., the Pharmacia Recombinant Phage Antibody System, catalog no.27-9400-01; and the Stratagene SurfZAP™ phage display kit, catalog no.240612). There are also other methods and reagents that can be used ingenerating and screening antibody display libraries (see, e.g., Ladneret al. U.S. Pat. No. 5,223,409; Kang et al. PCT Publication No. WO92/18619; Dower et al. PCT Publication No. WO 91/17271; Winter et al.PCT Publication No. WO 92/20791; Markland et al. PCT Publication No. WO92/15679; Breitling et al. PCT Publication No. WO 93/01288; McCaffertyet al. PCT Publication No. WO 92/01047; Garrard et al. PCT PublicationNo. WO 92/09690; Fuchs et al. (1991) Bio/Technology 9:1370-1372; Hay etal. (1992) Hum. Antibod. Hybridomas 3:81-85; Huse et al. (1989) Science246:1275-1281; McCafferty et al., Nature (1990) 348:552-554; Griffithset al. (1993) EMBO J 12:725-734; Hawkins et al. (1992) J. Mol. Biol.226:889-896; Clackson et al. (1991) Nature 352:624-628; Gram et al.(1992) Proc. Natl. Acad. Sci. USA 89:3576-3580; Garrad et al. (1991)Bio/Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res19:4133-4137; and Barbas et al. (1991) Proc. Natl. Acad. Sci. USA88:7978-7982.

In one embodiment, to isolate human antibodies specific for the targetantigen with the desired characteristics, a human VH and VL library arescreened to select for antibody fragments having the desiredspecificity. The antibody libraries used in this method are preferablyscFv libraries prepared and screened as described herein and in the art(McCafferty et al., PCT Publication No. WO 92/01047, McCafferty et al.,(Nature 348:552-554 (1990)); and Griffiths et al., (EMBO J 12:725-734(1993)). The scFv antibody libraries preferably are screened usingtarget protein as the antigen.

Alternatively, the Fd fragment (VH-CH1) and light chain (VL-CL) ofantibodies are separately cloned by PCR and recombined randomly incombinatorial phage display libraries, which can then be selected forbinding to a particular antigen. The Fab fragments are expressed on thephage surface, i.e., physically linked to the genes that encode them.Thus, selection of Fab by antigen binding co-selects for the Fabencoding sequences, which can be amplified subsequently. Through severalrounds of antigen binding and re-amplification, a procedure termedpanning, Fab specific for the antigen are enriched and finally isolated.

In 1994, an approach for the humanization of antibodies, called “guidedselection”, was described. Guided selection utilizes the power of thephage display technique for the humanization of mouse monoclonalantibody (See Jespers, L. S., et al., Bio/Technology 12, 899-903(1994)). For this, the Fd fragment of the mouse monoclonal antibody canbe displayed in combination with a human light chain library, and theresulting hybrid Fab library may then be selected with antigen. Themouse Fd fragment thereby provides a template to guide the selection.Subsequently, the selected human light chains are combined with a humanFd fragment library. Selection of the resulting library yields entirelyhuman Fab.

A variety of procedures have been described for deriving humanantibodies from phage-display libraries (See, for example, Hoogenboom etal., J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol,222:581-597 (1991); U.S. Pat. Nos. 5,565,332 and 5,573,905; Clackson,T., and Wells, J. A., TIBTECH 12, 173-184 (1994)). In particular, invitro selection and evolution of antibodies derived from phage displaylibraries has become a powerful tool (See Burton, D. R., and Barbas III,C. F., Adv. Immunol. 57, 191-280 (1994); Winter, G., et al., Annu. Rev.Immunol. 12, 433-455 (1994); U.S. patent publication no. 20020004215 andWO 92/01047; U.S. patent publication no. 20030190317; and U.S. Pat. Nos.6,054,287 and 5,877,293.

Watkins, “Screening of Phage-Expressed Antibody Libraries by CaptureLift,” Methods in Molecular Biology, Antibody Phage Display: Methods andProtocols 178:187-193 (2002), and U.S. patent publication no.20030044772, published Mar. 6, 2003, describe methods for screeningphage-expressed antibody libraries or other binding molecules by capturelift, a method involving immobilization of the candidate bindingmolecules on a solid support.

Fv fragments are displayed on the surface of phage, by the associationof one chain expressed as a phage protein fusion (e.g., with M13 geneIII) with the complementary chain expressed as a soluble fragment. It iscontemplated that the phage may be a filamentous phage such as one ofthe class I phages: fd, M13, f1, If1, lke, ZJ/Z, Ff and one of the classII phages Xf, Pf1 and Pf3. The phage may be M13, or fd or a derivativethereof.

Once initial human VL and VH segments are selected, “mix and match”experiments, in which different pairs of the initially selected VL andVH segments are screened for target binding, are performed to selectpreferred VL/VH pair combinations. Additionally, to further improve thequality of the antibody, the VL and VH segments of the preferred VL/VHpair(s) can be randomly mutated, preferably within the any of the CDR1,CDR2 or CDR3 region of VH and/or VL, in a process analogous to the invivo somatic mutation process responsible for affinity maturation ofantibodies during a natural immune response. This in vitro affinitymaturation can be accomplished by amplifying VL and VH regions using PCRprimers complimentary to the VH CDR1, CDR2, and CDR3, or VL CDR1, CDR2,and CDR3, respectively, which primers have been “spiked” with a randommixture of the four nucleotide bases at certain positions such that theresultant PCR products encode VL and VH segments into which randommutations have been introduced into the VH and/or VL CDR3 regions. Theserandomly mutated VL and VH segments can be rescreened for binding totarget antigen.

Following screening and isolation of an target specific antibody from arecombinant immunoglobulin display library, nucleic acid encoding theselected antibody can be recovered from the display package (e.g., fromthe phage genome) and subcloned into other expression vectors bystandard recombinant DNA techniques. If desired, the nucleic acid can befurther manipulated to create other antibody forms of the disclosure, asdescribed below. To express a recombinant human antibody isolated byscreening of a combinatorial library, the DNA encoding the antibody iscloned into a recombinant expression vector and introduced into amammalian host cell, as described herein.

It is contemplated that the phage display method may be carried out in amutator strain of bacteria or host cell. A mutator strain is a host cellwhich has a genetic defect which causes DNA replicated within it to bemutated with respect to its parent DNA. Example mutator strains areNR9046mutD5 and NR9046 mut T1.

It is also contemplated that the phage display method may be carried outusing a helper phage. This is a phage which is used to infect cellscontaining a defective phage genome and which functions to complementthe defect. The defective phage genome can be a phagemid or a phage withsome function encoding gene sequences removed. Examples of helper phagesare M13K07, M13K07 gene III no. 3; and phage displaying or encoding abinding molecule fused to a capsid protein.

Antibodies are also generated via phage display screening methods usingthe hierarchical dual combinatorial approach as disclosed in WO 92/01047in which an individual colony containing either an H or L chain clone isused to infect a complete library of clones encoding the other chain (Lor H) and the resulting two-chain specific binding member is selected inaccordance with phage display techniques such as those describedtherein. This technique is also disclosed in Marks et al,(Bio/Technology, 10:779-783 (1992)).

Methods for display of peptides on the surface of yeast, microbial andmammalian cells have also been used to identify antigen specificantibodies. See, for example, U.S. Pat. Nos. 5,348,867; 5,723,287;6,699,658; Wittrup, Curr Op. Biotech. 12:395-99 (2001); Lee et al,Trends in Biotech. 21(1) 45-52 (2003); Surgeeva et al, Adv. Drug Deliv.Rev. 58: 1622-54 (2006). Antibody libraries may be attached to yeastproteins, such as agglutinin, effectively mimicking the cell surfacedisplay of antibodies by B cells in the immune system.

In addition to phage display methods, antibodies may be isolated usingin vitro display methods and microbial cell display, including ribosomedisplay and mRNA display (Amstutz et al, Curr. Op. Biotech. 12: 400-05(2001)). Selection of polypeptides using ribosome display is describedin Hanes et al., (Proc. Natl Acad Sci USA, 94:4937-4942 (1997)) and U.S.Pat. Nos. 5,643,768 and 5,658,754 issued to Kawasaki. Ribosome displayis also useful for rapid large scale mutational analysis of antibodies.The selective mutagenesis approach also provides a method of producingantibodies with improved activities that can be selected using ribosomaldisplay techniques.

Amino Acid Sequence Variants

Modified polypeptide compositions comprising one, two, three, four,five, and/or six CDRs of an antibody may be generated, wherein a CDR isaltered to provide increased specificity or affinity to the targetmolecule. Sites within antibody CDRs are typically modified in series,e.g., by substituting first with conservative choices (e.g., hydrophobicamino acid substituted for a non-identical hydrophobic amino acid) andthen with more dissimilar choices (e.g., hydrophobic amino acidsubstituted for a charged amino acid), and then deletions or insertionsmay be made at the target site. For example, using the conservedframework sequences surrounding the CDRs, PCR primers complementary tothese consensus sequences are generated to amplify the antigen-specificCDR sequence located between the primer regions. Techniques for cloningand expressing nucleotide and polypeptide sequences are well-establishedin the art [see e.g. Sambrook et al., Molecular Cloning: A LaboratoryManual, 2nd Edition, Cold Spring Harbor, N.Y. (1989)]. The amplified CDRsequences are ligated into an appropriate plasmid. The plasmidcomprising one, two, three, four, five and/or six cloned CDRs optionallycontains additional polypeptide encoding regions linked to the CDR.

Modifications may be made by conservative or non-conservative amino acidsubstitutions described in greater detail below. “Insertions” or“deletions” are preferably in the range of about 1 to 20 amino acids,more preferably 1 to 10 amino acids. The variation may be introduced bysystematically making substitutions of amino acids in an antibodypolypeptide molecule using recombinant DNA techniques and assaying theresulting recombinant variants for activity. Nucleic acid alterationscan be made at sites that differ in the nucleic acids from differentspecies (variable positions) or in highly conserved regions (constantregions). Methods for altering antibody sequences and expressingantibody polypeptide compositions useful in the disclosure are describedin the art. See e.g., U.S. Pat. No. 8,569,462

As used herein, the term “salvage receptor binding epitope” refers to anepitope of the Fc region of an IgG molecule (e.g., IgG1, IgG2, IgG3, orIgG4) that is responsible for increasing the in vivo serum half-life ofthe IgG molecule.

Another type of variant is an amino acid substitution variant. Thesevariants have at least one amino acid residue in the antibody moleculeremoved and a different residue inserted in its place. Substitutionalmutagenesis within any of the hypervariable or CDR regions or frameworkregions is contemplated. Conservative substitutions involve replacing anamino acid with another member of its class. Non-conservativesubstitutions involve replacing a member of one of these classes with amember of another class.

Conservative amino acid substitutions are made on the basis ofsimilarity in polarity, charge, solubility, hydrophobicity,hydrophilicity, and/or the amphipathic nature of the residues involved.For example, nonpolar (hydrophobic) amino acids include alanine (Ala,A), leucine (Leu, L), isoleucine (Ile, I), valine (Val, V), proline(Pro, P), phenylalanine (Phe, F), tryptophan (Trp, W), and methionine(Met, M); polar neutral amino acids include glycine (Gly, G), serine(Ser, S), threonine (Thr, T), cysteine (Cys, C), tyrosine (Tyr, Y),asparagine (Asn, N), and glutamine (Gln, Q); positively charged (basic)amino acids include arginine (Arg, R), lysine (Lys, K), and histidine(His, H); and negatively charged (acidic) amino acids include asparticacid (Asp, D) and glutamic acid (Glu, E).

Any cysteine residue not involved in maintaining the proper conformationof the antibody also may be substituted, generally with serine, toimprove the oxidative stability of the molecule and prevent aberrantcrosslinking. Conversely, cysteine bond(s) may be added to the antibodyto improve its stability (particularly where the antibody is an antibodyfragment such as an Fv fragment).

Altered Glycosylation

Antibody variants can also be produced that have a modifiedglycosylation pattern relative to the parent antibody, for example,deleting one or more carbohydrate moieties found in the antibody, and/oradding one or more glycosylation sites that are not present in theantibody.

Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain. Thepresence of either of these tripeptide sequences in a polypeptidecreates a potential glycosylation site. Thus, N-linked glycosylationsites may be added to an antibody by altering the amino acid sequencesuch that it contains one or more of these tripeptide sequences.O-linked glycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used. O-linked glycosylation sites may beadded to an antibody by inserting or substituting one or more serine orthreonine residues to the sequence of the original antibody.

Fc glycans influence the binding of IgG to Fc receptors and C1q, and aretherefore important for IgG effector functions. Antibody variants withmodified Fc glycans and altered effector function may be produced. Forexample, antibodies with modified terminal sugars such as sialic acids,core fucose, bisecting N-acetylglucosamine, and mannose residues mayhave altered binding to the FcγRIIIa receptor and altered ADCC activity.In a further example, antibodies with modified terminal galactoseresidues may have altered binding to C1q and altered CDC activity (Raju,Curr. Opin. Immunol. 20: 471-78 (2008).

Also contemplated for use in the methods are antibody molecules withabsent or reduced fucosylation that exhibit improved ADCC activity. Avariety of ways are known in the art to accomplish this. For example,ADCC effector activity is mediated by binding of the antibody moleculeto the FcγRIII receptor, which has been shown to be dependent on thecarbohydrate structure of the N-linked glycosylation at the Asn-297 ofthe CH2 domain. Non-fucosylated antibodies bind this receptor withincreased affinity and trigger FcγRIII-mediated effector functions moreefficiently than native, fucosylated antibodies. For example,recombinant production of non-fucosylated antibody in CHO cells in whichthe alpha-1,6-fucosyl transferase enzyme has been knocked out results inantibody with 100-fold increased ADCC activity (Yamane-Ohnuki et al.,Biotechnol Bioeng. 87:614-22 (2004)). Similar effects can beaccomplished through decreasing the activity of this or other enzymes inthe fucosylation pathway, e.g., through siRNA or antisense RNAtreatment, engineering cell lines to knockout the enzyme(s), orculturing with selective glycosylation inhibitors (Rothman et al., MolImmunol. 26:1113-23 (1989)). Some host cell strains, e.g. Lec13 or rathybridoma YB2/0 cell line naturally produce antibodies with lowerfucosylation levels. (Shields et al., J Biol Chem. 277:26733-40 (2002);Shinkawa et al., J Biol Chem. 278:3466-73 (2003)). An increase in thelevel of bisected carbohydrate, e.g. through recombinantly producingantibody in cells that overexpress GnTIII enzyme, has also beendetermined to increase ADCC activity (Umana et al., Nat Biotechnol.17:176-80 (1999)). It has been predicted that the absence of only one ofthe two fucose residues may be sufficient to increase ADCC activity(Ferrara et al., Biotechnol Bioeng. 93:851-61 (2006)).

Variants with Altered Effector Function

Other modifications of the antibodies for use in the methods arecontemplated. In one aspect, it may be desirable to modify an antibodyused herein with respect to effector function, for example, to enhancethe effectiveness of the antibody in treating cancer. One method formodifying effector function teaches that cysteine residue(s) may beintroduced in the Fc region, thereby allowing interchain disulfide bondformation in this region. The homodimeric antibody thus generated mayhave improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). See Caron et al., (J. Exp Med. 176: 1191-1195(1992)) and Shopes, B. (J. Immunol. 148: 2918-2922 (1992)). Homodimericantibodies with enhanced anti-tumor activity may also be prepared usingheterobifunctional cross-linkers as described in Wolff et al., (CancerResearch 53: 2560-2565 (1993)). Alternatively, an antibody can beengineered which has dual Fc regions and may thereby have enhancedcomplement lysis and ADCC capabilities. See Stevenson et al.,(Anti-Cancer Drug Design 3: 219-230 (1989)). In addition, it has beenshown that sequences within the CDR can cause an antibody to bind to MHCClass II and trigger an unwanted helper T-cell response. A conservativesubstitution can allow the antibody to retain binding activity yet loseits ability to trigger an unwanted T-cell response.

In certain embodiments of the present disclosure, it may be desirable touse an antibody fragment, rather than an intact antibody, to increasetumor penetration, for example. In this case, it may be desirable tomodify the antibody fragment in order to increase its serum half-life,for example, adding molecules such as PEG or other water solublepolymers, including polysaccharide polymers, to antibody fragments toincrease the half-life.

The salvage receptor binding epitope preferably constitutes a regionwherein any one or more amino acid residues from one or two loops of aFc domain are transferred to an analogous position of the antibodyfragment. Even more preferably, three or more residues from one or twoloops of the Fc domain are transferred. Still more preferred, theepitope is taken from the CH2 domain of the Fc region (e.g., of an IgG)and transferred to the CH1, CH3, or VH region, or more than one suchregion, of the antibody. Alternatively, the epitope is taken from theCH2 domain of the Fc region and transferred to the CL region or VLregion, or both, of the antibody fragment.

Thus, antibodies of the present disclosure may comprise a human Fcportion, a human consensus Fc portion, or a variant thereof that retainsthe ability to interact with the Fc salvage receptor, including variantsin which cysteines involved in disulfide bonding are modified orremoved, and/or in which the a met is added at the N-terminus and/or oneor more of the N-terminal 20 amino acids are removed, and/or regionsthat interact with complement, such as the C1q binding site, areremoved, and/or the ADCC site is removed [see, e.g., Sarmay et al.,Molec. Immunol. 29:633-9 (1992)].

Shields et al. reported that IgG1 residues involved in binding to allhuman Fc receptors are located in the CH2 domain proximal to the hingeand fall into two categories as follows: 1) positions that may interactdirectly with all FcR include Leu234-Pro238, Ala327, and Pro329 (andpossibly Asp265); 2) positions that influence carbohydrate nature orposition include Asp265 and Asn297. The additional IgG1 residues thataffected binding to Fc receptor II are as follows: (largest effect)Arg255, Thr256, Glu258, Ser267, Asp270, Glu272, Asp280, Arg292, Ser298,and (less effect) His268, Asn276, His285, Asn286, Lys290, Gln295,Arg301, Thr307, Leu309, Asn315, Lys322, Lys326, Pro331, Ser337, Ala339,Ala378, and Lys414. A327Q, A327S, P329A, D265A and D270A reducedbinding. In addition to the residues identified above for all FcR,additional IgG1 residues that reduced binding to Fc receptor IIIA by 40%or more are as follows: Ser239, Ser267 (Gly only), His268, Glu293,Gln295, Tyr296, Arg301, Val303, Lys338, and Asp376. Variants thatimproved binding to FcRIIIA include T256A, K290A, S298A, E333A, K334A,and A339T. Lys414 showed a 40% reduction in binding for FcRIIA andFcRIIB, Arg416 a 30% reduction for FcRIIA and FcRIIIA, Gln419 a 30%reduction to FcRIIA and a 40% reduction to FcRIIB, and Lys360 a 23%improvement to FcRIIIA See also Presta et al., (Biochem. Soc. Trans.30:487-490, 2001), incorporated herein by reference in its entirety,which described several positions in the Fc region of IgG1 were foundwhich improved binding only to specific Fc gamma receptors (R) orsimultaneously improved binding to one type of Fc gamma R and reducedbinding to another type. Selected IgG1 variants with improved binding toFc gamma RIIIa were then tested in an in vitro antibody-dependentcellular cytotoxicity (ADCC) assay and showed an enhancement in ADCCwhen either peripheral blood mononuclear cells or natural killer cellswere used.

For example, U.S. Pat. No. 6,194,551, incorporated herein by referencein its entirety, describes variants with altered effector functioncontaining mutations in the human IgG Fc region, at amino acid position329, 331 or 322 (using Kabat numbering), some of which display reducedC1q binding or CDC activity. As another example, U.S. Pat. No.6,737,056, incorporated herein by reference in its entirety, describesvariants with altered effector or Fc-gamma-receptor binding containingmutations in the human IgG Fc region, at amino acid position 238, 239,248, 249, 252, 254, 255, 256, 258, 265, 267, 268, 269, 270, 272, 276,278, 280, 283, 285, 286, 289, 290, 292, 294, 295, 296, 298, 301, 303,305, 307, 309, 312, 315, 320, 322, 324, 326, 327, 329, 330, 331, 333,334, 335, 337, 338, 340, 360, 373, 376, 378, 382, 388, 389, 398, 414,416, 419, 430, 434, 435, 437, 438 or 439 (using Kabat numbering), someof which display receptor binding profiles associated with reduced ADCCor CDC activity. Of these, a mutation at amino acid position 238, 265,269, 270, 327 or 329 are stated to reduce binding to FcRI, a mutation atamino acid position 238, 265, 269, 270, 292, 294, 295, 298, 303, 324,327, 329, 333, 335, 338, 373, 376, 414, 416, 419, 435, 438 or 439 arestated to reduce binding to FcRII, and a mutation at amino acid position238, 239, 248, 249, 252, 254, 265, 268, 269, 270, 272, 278, 289, 293,294, 295, 296, 301, 303, 322, 327, 329, 338, 340, 373, 376, 382, 388,389, 416, 434, 435 or 437 is stated to reduce binding to FcRIII

U.S. Pat. No. 5,624,821, incorporated by reference herein in itsentirety, reports that Clq binding activity of an murine antibody can bealtered by mutating amino acid residue 318, 320 or 322 of the heavychain and that replacing residue 297 (Asn) results in removal of lyticactivity.

U.S. Patent Publication No. 20040132101, incorporated by referenceherein in its entirety, describes variants with mutations at amino acidpositions 240, 244, 245, 247, 262, 263, 266, 299, 313, 325, 328, or 332(using Kabat numbering) or positions 234, 235, 239, 240, 241, 243, 244,245, 247, 262, 263, 264, 265, 266, 267, 269, 296, 297, 298, 299, 313,325, 327, 328, 329, 330, or 332 (using Kabat numbering), of whichmutations at positions 234, 235, 239, 240, 241, 243, 244, 245, 247, 262,263, 264, 265, 266, 267, 269, 296, 297, 298, 299, 313, 325, 327, 328,329, 330, or 332 may reduce ADCC activity or reduce binding to an Fcgamma receptor.

Covalent Modifications

Antibodies comprising covalent modifications are also contemplated foruse in the methods. They may be made by chemical synthesis or byenzymatic or chemical cleavage of the antibody, if applicable. Othertypes of covalent modifications of the antibody are introduced into themolecule by reacting targeted amino acid residues of the antibody withan organic derivatizing agent that is capable of reacting with selectedside chains or the N- or C-terminal residues.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Other modifications include histidlyl, lysinyl arginyl, tyrosyl,glutaminyl and asparaginyl hydroxylation of proline and lysine. Methodsfor making such modifications are disclosed in U.S. Pat. No. 8,926,976,incorporated herein by reference, and in the art.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R-N.dbd.C.dbd.N-R′), where R and R′ aredifferent alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)),acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

Another type of covalent modification involves chemically orenzymatically coupling glycosides to the antibody. These procedures areadvantageous in that they do not require production of the antibody in ahost cell that has glycosylation capabilities for N- or O-linkedglycosylation. Depending on the coupling mode used, the sugar(s) may beattached to (a) arginine and histidine, (b) free carboxyl groups, (c)free sulfhydryl groups such as those of cysteine, (d) free hydroxylgroups such as those of serine, threonine, or hydroxyproline, (e)aromatic residues such as those of phenylalanine, tyrosine, ortryptophan, or (f) the amide group of glutamine. These methods aredescribed in WO87/05330 and in Aplin and Wriston, (CRC Crit. Rev.Biochem., pp. 259-306 (1981)).

Removal of any carbohydrate moieties present on the antibody may beaccomplished chemically or enzymatically. Chemical deglycosylationrequires exposure of the antibody to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving theantibody intact. Chemical deglycosylation is described by Hakimuddin, etal., (Arch. Biochem. Biophys. 259: 52 (1987)) and by Edge et al., (Anal.Biochem. 118: 131 (1981)). Enzymatic cleavage of carbohydrate moietieson antibodies can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., (Meth. Enzymol. 138:350 (1987)).

Another type of covalent modification of the antibody comprises linkingthe antibody to one of a variety of nonproteinaceous polymers, e.g.,polyethylene glycol, polypropylene glycol, polyoxyethylated polyols,polyoxyethylated sorbitol, polyoxyethylated glucose, polyoxyethylatedglycerol, polyoxyalkylenes, or polysaccharide polymers such as dextran.Such methods are known in the art.

Derivatives

As stated above, derivative, when used in connection with antibodysubstances and polypeptides, refers to polypeptides chemically modifiedby such techniques as ubiquitination, labeling (e.g., with radionuclidesor various enzymes), covalent polymer attachment such as PEGylation(derivatization with polyethylene glycol) and insertion or substitutionby chemical synthesis of amino acids such as ornithine. Derivatives ofthe antibodies disclosed herein are also useful as therapeutic agentsand may be used in the methods herein.

The conjugated moiety can be incorporated in or attached to an antibodysubstance either covalently, or through ionic, van der Waals or hydrogenbonds, e.g., incorporation of radioactive nucleotides, or biotinylatednucleotides that are recognized by streptavadin.

Polyethylene glycol (PEG) may be attached to the antibody substances toprovide a longer half-life in vivo. The PEG group may be of anyconvenient molecular weight and may be linear or branched. The averagemolecular weight of the PEG will preferably range from about 2kiloDalton (“kD”) to about 100 kDa, more preferably from about 5 kDa toabout 50 kDa, most preferably from about 5 kDa to about 10 kDa. The PEGgroups will generally be attached to the antibody substances of thedisclosure via acylation or reductive alkylation through a natural orengineered reactive group on the PEG moiety (e.g., an aldehyde, amino,thiol, or ester group) to a reactive group on the antibody substance(e.g., an aldehyde, amino, or ester group). Addition of PEG moieties toantibody substances can be carried out using techniques well-known inthe art. See, e.g., International Publication No. WO 96/11953 and U.S.Pat. No. 4,179,337.

Ligation of the antibody substance with PEG usually takes place inaqueous phase and can be easily monitored by reverse phase analyticalHPLC. The PEGylated substances are purified by preparative HPLC andcharacterized by analytical HPLC, amino acid analysis and laserdesorption mass spectrometry.

Antibody Conjugates

An antibody may be administered in its “naked” or unconjugated form, ormay be conjugated directly to other therapeutic or diagnostic agents, ormay be conjugated indirectly to carrier polymers comprising such othertherapeutic or diagnostic agents. In some embodiments the antibody isconjugated to a cytotoxic agent such as a chemotherapeutic agent, adrug, a growth inhibitory agent, a toxin (e.g., an enzymatically activetoxin of bacterial, fungal, plant, or animal origin, or fragmentsthereof), or a radioactive isotope (i.e., a radioconjugate). Suitablechemotherapeutic agents include: daunomycin, doxorubicin, methotrexate,and vindesine (Rowland et al., (1986) supra). Suitable toxins include:bacterial toxins such as diphtheria toxin; plant toxins such as ricin;small molecule toxins such as geldanamycin (Mandler et al J. Natl.Cancer Inst. 92(19):1573-81 (2000); Mandler et al., Bioorg. Med. Chem.Letters 10:1025-1028 (2000); Mandler et al., Bioconjugate Chem.13.786-91 (2002)), maytansinoids (EP 1391213; Liu et al., Proc. Natl.Acad. Sci. USA 93:8618-23 (1996)), auristatins (Doronina et al., Nat.Biotech. 21: 778-84 (2003) and calicheamicin (Lode et al., Cancer Res.58:2928 (1998); Hinman et al., Cancer Res. 53:3336-3342 (1993)).Antibody-Drug Conjugates and methods are reviewed in Ducry L, mAbs.6(1), 2014 and Shen W C, AAPS., 17: 3-7, 2015.

Antibodies can be detectably labeled through the use of radioisotopes,affinity labels (such as biotin, avidin, etc.), enzymatic labels (suchas horseradish peroxidase, alkaline phosphatase, etc.) fluorescent orluminescent or bioluminescent labels (such as FITC or rhodamine, etc.),paramagnetic atoms, and the like. Procedures for accomplishing suchlabeling are well known in the art; for example, see (Sternberger, L. A.et al., J. Histochem. Cytochem. 18:315 (1970); Bayer, E. A. et al.,Meth. Enzym. 62:308 (1979); Engval, E. et al., Immunol. 109:129 (1972);Goding, J. W. J. Immunol. Meth. 13:215 (1976)).

Conjugation of antibody moieties is described in U.S. Pat. No.6,306,393. General techniques are also described in Shih et al., Int. J.Cancer 41:832-839 (1988); Shih et al., Int. J. Cancer 46:1101-1106(1990); and Shih et al., U.S. Pat. No. 5,057,313. This general methodinvolves reacting an antibody component having an oxidized carbohydrateportion with a carrier polymer that has at least one free amine functionand that is loaded with a plurality of drug, toxin, chelator, boronaddends, or other therapeutic agent. This reaction results in an initialSchiff base (imine) linkage, which can be stabilized by reduction to asecondary amine to form the final conjugate.

The carrier polymer may be, for example, an aminodextran or polypeptideof at least 50 amino acid residues. Various techniques for conjugating adrug or other agent to the carrier polymer are known in the art. Apolypeptide carrier can be used instead of aminodextran, but thepolypeptide carrier should have at least 50 amino acid residues in thechain, preferably 100-5000 amino acid residues. At least some of theamino acids should be lysine residues or glutamate or aspartateresidues. The pendant amines of lysine residues and pendant carboxylatesof glutamine and aspartate are convenient for attaching a drug, toxin,immunomodulator, chelator, boron addend or other therapeutic agent.Examples of suitable polypeptide carriers include polylysine,polyglutamic acid, polyaspartic acid, co-polymers thereof, and mixedpolymers of these amino acids and others, e.g., serines, to conferdesirable solubility properties on the resultant loaded carrier andconjugate. Examples of agents to which the antibody can be conjugatedinclude any of the cytotoxic or chemotherapeutic agents describedherein.

Alternatively, conjugated antibodies can be prepared by directlyconjugating an antibody component with a therapeutic agent. The generalprocedure is analogous to the indirect method of conjugation except thata therapeutic agent is directly attached to an oxidized antibodycomponent. For example, a carbohydrate moiety of an antibody can beattached to polyethyleneglycol to extend half-life.

Alternatively, a therapeutic agent can be attached at the hinge regionof a reduced antibody component via disulfide bond formation, or using aheterobifunctional cross-linker, such as N-succinyl3-(2-pyridyldithio)proprionate (SPDP). Yu et al., Int. J. Cancer 56:244(1994). General techniques for such conjugation are well-known in theart. See, for example, Wong, Chemistry Of Protein Conjugation andCross-Linking (CRC Press 1991); Upeslacis et al., “Modification ofAntibodies by Chemical Methods,” in Monoclonal Antibodies: Principlesand Applications, Birch et al. (eds.), pages 187-230 (Wiley-Liss, Inc.1995); Price, “Production and Characterization of SyntheticPeptide-Derived Antibodies,” in Monoclonal Antibodies: Production,Engineering and Clinical Application, Ritter et al. (eds.), pages 60-84(Cambridge University Press 1995). A variety of bifunctional proteincoupling agents are known in the art, such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene).

Antibody Fusion Proteins

Methods of making antibody-toxin fusion proteins in which a recombinantmolecule comprises one or more antibody components and a toxin orchemotherapeutic agent also are known to those of skill in the art. Forexample, antibody-Pseudomonas exotoxin A fusion proteins have beendescribed by Chaudhary et al., Nature 339:394 (1989), Brinkmann et al.,Proc. Nat'l Acad. Sci. USA 88:8616 (1991), Batra et al., Proc. Nat'lAcad. Sci. USA 89:5867 (1992), Friedman et al., J. Immunol. 150:3054(1993), Wels et al., Int. J. Can. 60:137 (1995), Fominaya et al., J.Biol. Chem. 271:10560 (1996), Kuan et al., Biochemistry 35:2872 (1996),and Schmidt et al., Int. J. Can. 65:538 (1996). Antibody-toxin fusionproteins containing a diphtheria toxin moiety have been described byKreitman et al., Leukemia 7:553 (1993), Nicholls et al., J. Biol. Chem.268:5302 (1993), Thompson et al., J. Biol. Chem. 270:28037 (1995), andVallera et al., Blood 88:2342 (1996). Deonarain et al., Tumor Targeting1:177 (1995), have described an antibody-toxin fusion protein having anRNase moiety, while Linardou et al., Cell Biophys. 24-25:243 (1994),produced an antibody-toxin fusion protein comprising a DNase Icomponent. Gelonin was used as the toxin moiety in the antibody-toxinfusion protein of Wang et al., Abstracts of the 209th ACS NationalMeeting, Anaheim, Calif., Apr. 2-6, 1995, Part 1, BIOT005. As a furtherexample, Dohlsten et al., Proc. Nat'l Acad. Sci. USA 91:8945 (1994),reported an antibody-toxin fusion protein comprising Staphylococcalenterotoxin-A.

Illustrative of toxins which are suitably employed in the preparation ofsuch fusion proteins are ricin, abrin, ribonuclease, DNase I,Staphylococcal enterotoxin-A, pokeweed antiviral protein, gelonin,diphtherin toxin, Pseudomonas exotoxin, and Pseudomonas endotoxin. See,for example, Pastan et al., Cell 47:641 (1986), and Goldenberg, CA—ACancer Journal for Clinicians 44:43 (1994). Other suitable toxins areknown to those of skill in the art.

Antibodies of the present disclosure may also be used in ADEPT byconjugating the antibody to a prodrug-activating enzyme which converts aprodrug (e.g., a peptidyl chemotherapeutic agent, See WO81/01145) to anactive anti-cancer drug. See, for example, WO88/07378 and U.S. Pat. No.4,975,278.

The enzyme component of the immunoconjugate useful for ADEPT includesany enzyme capable of acting on a prodrug in such a way so as to convertit into its more active, cytotoxic form.

Enzymes that are useful in the present disclosure include, but are notlimited to, alkaline phosphatase useful for convertingphosphate-containing prodrugs into free drugs; arylsulfatase useful forconverting sulfate-containing prodrugs into free drugs; cytosinedeaminase useful for converting non-toxic 5-fluorocytosine into theanti-cancer drug, 5-fluorouracil; proteases, such as serratia protease,thermolysin, subtilisin, carboxypeptidases and cathepsins (such ascathepsins B and L), that are useful for converting peptide-containingprodrugs into free drugs; D-alanylcarboxypeptidases, useful forconverting prodrugs that contain D-amino acid substituents;carbohydrate-cleaving enzymes such as α-galactosidase and neuraminidaseuseful for converting glycosylated prodrugs into free drugs; β-lactamaseuseful for converting drugs derivatized with β-lactams into free drugs;and penicillin amidases, such as penicillin V amidase or penicillin Gamidase, useful for converting drugs derivatized at their aminenitrogens with phenoxyacetyl or phenylacetyl groups, respectively, intofree drugs. Alternatively, antibodies with enzymatic activity, alsoknown in the art as abzymes, can be used to convert the prodrugs of thedisclosure into free active drugs (See, e.g., Massey, Nature 328:457-458 (1987). Antibody-abzyme conjugates can be prepared as describedherein for delivery of the abzyme to a tumor cell population.

The enzymes above can be covalently bound to the antibodies bytechniques well known in the art such as the use of theheterobifunctional crosslinking reagents discussed above. Alternatively,fusion proteins comprising at least the antigen binding region of anantibody of the disclosure linked to at least a functionally activeportion of an enzyme of the disclosure can be constructed usingrecombinant DNA techniques well known in the art (See, e.g., Neubergeret al., Nature 312:604-608 (1984)).

Recombinant Production of Antibodies

DNA encoding an antibody described herein may be isolated and sequencedusing conventional procedures (e.g., by using oligonucleotide probesthat are capable of binding specifically to genes encoding the heavy andlight chains of the antibodies). Usually this requires cloning the DNAor, preferably, mRNA (i.e., cDNA) encoding the antibodies. Cloning andsequencing is carried out using standard techniques, such as for examplepolymerase chain reaction (PCR), (see, e.g., Sambrook et al. (1989)Molecular Cloning: A Laboratory Guide, Vols 1-3, Cold Spring HarborPress; Ausubel, et al. (Eds.), Protocols in Molecular Biology, JohnWiley & Sons (1994)), which are incorporated herein by reference).

Sequencing is carried out using standard techniques (see, e.g., Sambrooket al. (1989) Molecular Cloning: A Laboratory Guide, Vols 1-3, ColdSpring Harbor Press, and Sanger, F. et al. (1977) Proc. Natl. Acad. Sci.USA 74: 5463-5467, which is incorporated herein by reference). Bycomparing the sequence of the cloned nucleic acid with publishedsequences of human immunoglobulin genes and cDNAs, one of skill willreadily be able to determine, depending on the region sequenced, (i) thegermline segment usage of the immunoglobulin polypeptide (including theisotype of the heavy chain) and (ii) the sequence of the heavy and lightchain variable regions, including sequences resulting from N-regionaddition and the process of somatic mutation. One source ofimmunoglobulin gene sequence information is the National Center forBiotechnology Information, National Library of Medicine, NationalInstitutes of Health, Bethesda, Md.

Once isolated, the DNA may be placed into expression vectors, which arethen transfected into host cells such as E. coli cells, simian COScells, human embryonic kidney 293 cells (e.g., 293E cells), Chinesehamster ovary (CHO) cells, or myeloma cells that do not otherwiseproduce immunoglobulin protein, to obtain the synthesis of monoclonalantibodies in the recombinant host cells. Recombinant production ofantibodies is well known in the art.

In an alternative embodiment, the amino acid sequence of animmunoglobulin of interest may be determined by direct proteinsequencing. Suitable encoding nucleotide sequences can be designedaccording to a universal codon table.

For recombinant production of the antibodies, the nucleic acid encodingit is isolated and inserted into a replicable vector for further cloning(amplification of the DNA) or for expression. DNA encoding themonoclonal antibody is readily isolated and sequenced using conventionalprocedures (e.g., by using oligonucleotide probes that are capable ofbinding specifically to genes encoding the heavy and light chains of theantibody). Many vectors are available. The vector components generallyinclude, but are not limited to, one or more of the following: a signalsequence, an origin of replication, one or more selective marker genes,an enhancer element, a promoter, and a transcription terminationsequence, which are known and described in the art.

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41 Pdisclosed in DD 266,710 published Apr. 12, 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. One preferred E. coli cloning host is E.coli 294 (ATCC 31,446), although other strains such as E. coli B, E.coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.These examples are illustrative rather than limiting.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans,and K. marxianus; yarrowia (EP 402,226); Pichia pastors (EP 183,070);Candida; Trichoderma reesia (EP 244,234); Neurospora crassa;Schwanniomyces such as Schwanniomyces occidentalis; and filamentousfungi such as, e.g., Neurospora, Penicillium, Tolypocladium, andAspergillus hosts such as A. nidulans and A. niger.

Suitable host cells for the expression of glycosylated antibody arederived from multicellular organisms. Examples of invertebrate cellsinclude plant and insect cells. Numerous baculoviral strains andvariants and corresponding permissive insect host cells from hosts suchas Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedesalbopictus (mosquito), Drosophila melanogaster (fruitfly), and Bombyxmori have been identified. A variety of viral strains for transfectionare publicly available, e.g., the L-1 variant of Autographa californicaNPV and the Bm-5 strain of Bombyx mori NPV, and such viruses may be usedas the virus herein according to the present disclosure, particularlyfor transfection of Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,tobacco, lemna, and other plant cells can also be utilized as hosts.

Examples of useful mammalian host cell lines are Chinese hamster ovarycells, including CHOK1 cells (ATCC CCL61), DXB-11, DG-44, and Chinesehamster ovary cells/−DHFR (CHO, Urlaub et al., Proc. Natl. Acad. Sci.USA 77: 4216 (1980)); monkey kidney CV1 line transformed by SV40 (COS-7,ATCC CRL 1651); human embryonic kidney line (293 or 293 cells subclonedfor growth in suspension culture, (Graham et al., J. Gen Virol. 36: 59,1977); baby hamster kidney cells (BHK, ATCC CCL 10); mouse sertoli cells(TM4, Mather, (Biol. Reprod. 23: 243-251, 1980); monkey kidney cells(CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCCCRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); caninekidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCCCRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (HepG2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells(Mather et al., Annals N.Y. Acad. Sci. 383: 44-68 (1982)); MRC 5 cells;FS4 cells; and a human hepatoma line (Hep G2).

Host cells are transformed or transfected with the above-describedexpression or cloning vectors for antibody production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences. In addition, novel vectors and transfected cell lineswith multiple copies of transcription units separated by a selectivemarker are particularly useful and preferred for the expression ofantibodies that bind target.

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium, including from microbial cultures. If the antibody is producedintracellularly, as a first step, the particulate debris, either hostcells or lysed fragments, is removed, for example, by centrifugation orultrafiltration. Better et al. (Science 240:1041-43, 1988; ICSU ShortReports 10:105 (1990); and Proc. Natl. Acad. Sci. USA 90:457-461 (1993)describe a procedure for isolating antibodies which are secreted to theperiplasmic space of E. coli. [See also, (Carter et al., Bio/Technology10:163-167 (1992)].

The antibody composition prepared from microbial or mammalian cells canbe purified using, for example, hydroxylapatite chromatography cation oravian exchange chromatography, and affinity chromatography, withaffinity chromatography being the preferred purification technique. Thesuitability of protein A as an affinity ligand depends on the speciesand isotype of any immunoglobulin Fc domain that is present in theantibody. Protein A can be used to purify antibodies that are based onhuman γ1, γ2, or γ4 heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13, 1983). Protein G is recommended for all mouse isotypes and forhuman γ3 (Guss et al., EMBO J. 5:15671575 (1986)). The matrix to whichthe affinity ligand is attached is most often agarose, but othermatrices are available. Mechanically stable matrices such as controlledpore glass or poly(styrenedivinyl)benzene allow for faster flow ratesand shorter processing times than can be achieved with agarose. Wherethe antibody comprises a CH 3 domain, the Bakerbond ABX™ resin (J. T.Baker, Phillipsburg, N.J.) is useful for purification. Other techniquesfor protein purification such as fractionation on an ion-exchangecolumn, ethanol precipitation, Reverse Phase HPLC, chromatography onsilica, chromatography on heparin SEPHAROSE® chromatography on an anionor cation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

Screening Methods

Effective therapeutics depend on identifying efficacious agents devoidof significant toxicity. Antibodies may be screened for binding affinityby methods known in the art. For example, gel-shift assays, Westernblots, radiolabeled competition assay, co-fractionation bychromatography, co-precipitation, cross linking, ELISA, and the like maybe used, which are described in, for example, Current Protocols inMolecular Biology (1999) John Wiley & Sons, NY, which is incorporatedherein by reference in its entirety.

Methods for assessing neutralizing biological activity of TGFβ andanti-TGFβ antibodies are known in the art. See, e.g., U.S. Pat. No.7,867,496. Examples of in vitro bioassays include: (1) induction ofcolony formation of NRK cells in soft agar in the presence of EGF(Roberts et al. (1981) Proc. Natl. Acad. Sci. USA, 78:5339-5343); (2)induction of differentiation of primitive mesenchymal cells to express acartilaginous phenotype (Seyedin et al. (1985) Proc. Natl. Acad. Sci.USA, 82:2267-2271); (3) inhibition of growth of Mv1Lu mink lungepithelial cells (Danielpour et al. (1989) J. Cell. Physiol., 138:79-86)and BBC-1 monkey kidney cells (Holley et al. (1980) Proc. Natl. Acad.Sci. USA, 77:5989-5992); (4) inhibition of mitogenesis of C3H/HeJ mousethymocytes (Wrann et al. (1987) EMBO J., 6:1633-1636); (5) inhibition ofdifferentiation of rat L6 myoblast cells (Florini et al. (1986) J. Biol.Chem., 261:16509-16513); (6) measurement of fibronectin production(Wrana et al. (1992) Cell, 71:1003-1014); (7) induction of plasminogenactivator inhibitor I (PAI-1) promoter fused to a luciferase reportergene (Abe et al. (1994) Anal. Biochem., 216:276-284); (8) sandwichenzyme-linked immunosorbent assays (Danielpour et al. (1989) GrowthFactors, 2:61-71); and (9) cellular assays described in Singh et al.(2003) Bioorg. Med. Chem. Lett., 13(24):4355-4359.

In some embodiments, antibody neutralization of TGFβ1 and TGFβ2 is atleast 2-50 fold, 10-100 fold, 2-fold, 5-fold, 10-fold, 25-fold, 50-foldor 100-fold, or 20-50%, 50-100%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%,90% or 100% more potent that neutralization of TGFβ3.

Additional methods for assessing the biological activity andneutralization of TGFβ (e.g., by TGFβ antibodies) are known in the art.For example, neutralization can be measured by neutralization assays andexpressed as an IC50 value. The IC50 value can be calculated for a givenmolecule by determining the concentration of molecule needed to elicithalf inhibition of the maximum biological response of a second moleculeor cell activity. The lower the IC50, the greater the potency of themolecule to inhibit the desired protein activity. Exemplaryneutralization assays contemplated herein include, but are not limitedto, an interleukin-11 release assay and an HT-2/IL-4 cell proliferationassay. In addition, a TGFβ activity assay can be carried out todetermine if the antibody inhibits one TGFβ isoform preferentially,including a pSMAD phosphorylation assay or an rhLAP binding assay.

Methods for assessing neutralizing biological activity of PD-1inhibitors and anti-PD-1 antibodies are known in the art. For example,neutralization can be measured by neutralization assays and expressed asan IC50 value. The IC50 value can be calculated for a given molecule bydetermining the concentration of molecule needed to elicit halfinhibition of the maximum biological response of a second molecule orcell activity. The lower the IC50, the greater the potency of themolecule to inhibit the desired protein activity. Exemplaryneutralization assays contemplated herein include, but are not limitedto, measuring the PD-1 antibodies ability to promote T-cell responses inhuman T cells, IFNγ release assay, or interleukin-2 secretion assay(Wang et al., Cancer Immunol Res., 2(9): 846-56 (2014).

Combination Therapy

A TGFβ inhibitor of the present disclosure is administered with a secondagent that inhibits PD-1 and the combination is useful to treat adisease or disorder as described herein. In the case of the use ofantibodies to inhibit TGFβ and PD-1, if more than one TGFβ antibody orPD-1 antibody is effective at binding to respective target antigens, itis contemplated that two or more antibodies to different epitopes of thetarget antigen and/or which bind preferentially to different isoforms ofTGFβ or PD-1 may be mixed such that the combination of antibodies, threeor four or more together provide still further improved efficacy againsta condition or disorder to be treated with inhibitors of TGFβ and PD-1.Compositions comprising one or more antibody of the invention may beadministered to persons or mammals suffering from, or predisposed tosuffer from, a condition or disorder associated with the targetpolypeptide of either TGFβ or PD-1.

Concurrent administration of two therapeutic agents does not requirethat the agents be administered at the same time or by the same route,as long as there is an overlap in the time period during which theagents are exerting their therapeutic effect. Simultaneous or sequentialadministration is contemplated, as is administration on different daysor weeks.

A third agent may also be used with an inhibitor of TGFβ and aninhibitor PD-1. The third agent may be other therapeutic agents, such ascytokines, growth factors, other inhibitors and antibodies to othertarget antigens, for example ipilimumab (YERVOY®, Bristol-Myers SquibbCompany), an antibody to CTLA-4; bevacizumab (AVASTIN®, Genentech), anantibody to VEGF-A; erlotinib (TARCEVA®, Genentech and OSIPharmaceuticals), a tyrosine kinase inhibitor which acts on EGFR,dasatinib (SPRYCEL®, Bristol-Myers Squibb Company), an oral Bcr-Abltyrosone kinase inhibitor; IL-21; pegylated IFN-α2b; axitinib (INLYTA®,Pfizer, Inc.), a tyrosine kinase inhibitor; and trametinib (MEKINIST®,GlaxoSmithKline), a MEK inhibitor (Philips and Atkins, Int Immunol.,27(1):39-46 (2015) which is incorporated herein by reference).

If the cancer is V600 mutation positive, as is the case for somecancers, in particular in melanoma (Ascierto P et al., J Transl Med.,10: 85, 2012) it is also contemplated that the third agent is BRAFinhibitor, for example vemurafenib or dabrafenib.

It is contemplated the inhibitors, such as antibodies, of the presentdisclosure may be given simultaneously, in the same formulation. It isfurther contemplated that the inhibitors are administered in a separateformulation and administered concurrently, with concurrently referringto agents given within 30 minutes of each other. It is furthercontemplated that the third agent may be given simultaneously with theinhibitors.

In another aspect, a TGFβ or a PD-1 inhibitor is administered prior toadministration of the other inhibitor composition. Prior administrationrefers to administration of an inhibitor within the range of one weekprior to treatment with the other inhibitor, up to 30 minutes beforeadministration of the other inhibitor. It is further contemplated thatan inhibitor is administered subsequent to administration of anotherinhibitor composition. Subsequent administration is meant to describeadministration from 30 minutes after antibody treatment up to one weekafter antibody administration. It is further contemplated that a thirdagent maybe administered in this manner prior to either the TGFβinhibitor or PD-1 inhibitor.

It is further contemplated that other adjunct therapies may beadministered, where appropriate. For example, the patient may also beadministered surgical therapy, chemotherapy, a cytotoxic agent,photodynamic therapy or radiation therapy where appropriate.

It is further contemplated that when the inhibitors or antibodies hereinare administered in combination with a third agent, such as for example,wherein the third agent is a cytokine or growth factor, or achemotherapeutic agent, the administration also includes use of aradiotherapeutic agent or radiation therapy. The radiation therapyadministered in combination with an antibody composition is administeredas determined by the treating physician, and at doses typically given topatients being treated for cancer.

A cytotoxic agent refers to a substance that inhibits or prevents thefunction of cells and/or causes destruction of cells. The term isintended to include radioactive isotopes (e.g., I131, I125, Y90 andRe186), chemotherapeutic agents, and toxins such as enzymatically activetoxins of bacterial, fungal, plant or animal origin or synthetic toxins,or fragments thereof. A non-cytotoxic agent refers to a substance thatdoes not inhibit or prevent the function of cells and/or does not causedestruction of cells. A non-cytotoxic agent may include an agent thatcan be activated to be cytotoxic. A non-cytotoxic agent may include abead, liposome, matrix or particle (see, e.g., U.S. Patent Publications2003/0028071 and 2003/0032995 which are incorporated by referenceherein). Such agents may be conjugated, coupled, linked or associatedwith an antibody according to the disclosure.

Chemotherapeutic agents contemplated for use with the antibodies of thepresent disclosure include, but are not limited to those listed in TableI:

TABLE I Alkylating agents Nitrogen mustards mechlorethaminecyclophosphamide ifosfamide melphalan chlorambucil Nitrosoureascarmustine (BCNU) lomustine (CCNU) semustine (methyl-CCNU)Ethylenimine/Methyl-melamine thriethylenemelamine (TEM) triethylenethiophosphoramide (thiotepa) hexamethylmelamine (HMM, altretamine) Alkylsulfonates busulfan Triazines dacarbazine (DTIC) Antimetabolites FolicAcid analogs methotrexate Trimetrexate Pemetrexed (Multi-targetedantifolate) Pyrimidine analogs 5-fluorouracil fluorodeoxyuridinegemcitabine cytosine arabinoside (AraC, cytarabine) 5-azacytidine2,2′-difluorodeoxy-cytidine Purine analogs 6-mercaptopurine6-thioguanine azathioprine 2′-deoxycoformycin (pentostatin)erythrohydroxynonyl-adenine (EHNA) fludarabine phosphate2-chlorodeoxyadenosine (cladribine, 2-CdA) Type I TopoisomeraseInhibitors camptothecin topotecan irinotecan Biological responsemodifiers G-CSF GM-CSF Differentiation Agents retinoic acid derivativesHormones and antagonists Adrenocorticosteroids/antagonists prednisoneand equivalents dexamethasone ainoglutethimide Progestinshydroxyprogesterone caproate medroxyprogesterone acetate megestrolacetate Estrogens diethylstilbestrol ethynyl estradiol/equivalentsAntiestrogen tamoxifen Androgens testosterone propionatefluoxymesterone/equivalents Antiandrogens flutamidegonadotropin-releasing hormone analogs leuprolide Nonsteroidalantiandrogens flutamide Natural products Antimitotic drugs Taxanespaclitaxel Vinca alkaloids vinblastine (VLB) vincristine vinorelbineTaxotere ® (docetaxel) estramustine estramustine phosphateEpipodophylotoxins etoposide teniposide Antibiotics actimomycin Ddaunomycin (rubido-mycin) doxorubicin (adria-mycin)mitoxantroneidarubicin bleomycin splicamycin (mithramycin) mitomycinCdactinomycin aphidicolin Enzymes L-asparaginase L-arginaseRadiosensitizers metronidazole misonidazole desmethylmisonidazolepimonidazole etanidazole nimorazole RSU 1069 EO9 RB 6145 SR4233nicotinamide 5-bromodeozyuridine 5-iododeoxyuridine bromodeoxycytidineMiscellaneous agents Platinium coordination complexes cisplatinCarboplatin oxaliplatin Anthracenedione mitoxantrone Substituted ureahydroxyurea Methylhydrazine derivatives N-methylhydrazine (MIH)procarbazine Adrenocortical suppressant mitotane (o,p′-DDD)ainoglutethimide Cytokines interferon (α, β, γ) interleukin-2Photosensitizers hematoporphyrin derivatives Photofrin ® benzoporphyrinderivatives Npe6 tin etioporphyrin (SnET2) pheoboride-abacteriochlorophyll-a naphthalocyanines phthalocyanines zincphthalocyanines Radiation X-ray ultraviolet light gamma radiationvisible light infrared radiation microwave radiation

Treatment of Disorders

In another embodiment, any of the types of inhibitors described hereinmay be used in the methods. In exemplary embodiments, the targetspecific antibody is a human, chimeric or humanized antibody. In anotherexemplary embodiment, the target is human and the patient is a humanpatient. Alternatively, the patient may be a mammal that expresses atarget protein that target specific antibody cross-reacts with. Theantibody may be administered to a non-human mammal expressing a targetprotein with which the antibody cross-reacts (i.e. a primate) forveterinary purposes or as an animal model of human disease. Such animalmodels may be useful for evaluating the therapeutic efficacy of targetspecific antibodies of the disclosure (Huang and Balmain, Cold SpringHarb Perspect Med., 4(9):a013623, 2014).

In one embodiment, the disclosure provides a method for treating canceror preventing the recurrence of cancer comprising administering to asubject in need thereof a therapeutically effective amount of a TGFβinhibitor and a PD-1 inhibitor or a pharmaceutical compositioncomprising one or both of the inhibitors as described herein.

Exemplary conditions or disorders that can be treated with inhibitors ofTGFβ and of PD-1 (e.g., antibodies of the present disclosure) includecancers, such as esophageal cancer, pancreatic cancer, metastaticpancreatic cancer, metastatic adenocarcinoma of the pancreas, bladdercancer, stomach cancer, fibrotic cancer, glioma, malignant glioma,diffuse intrinsic pontine glioma, recurrent childhood brain neoplasmrenal cell carcinoma, clear-cell metastatic renal cell carcinoma, kidneycancer, prostate cancer, metastatic castration resistant prostatecancer, stage IV prostate cancer, metastatic melanoma, melanoma,malignant melanoma, recurrent melanoma of the skin, melanoma brainmetastases, stage IIIA skin melanoma; stage IIIB skin melanoma, stageIIIC skin melanoma; stage IV skin melanoma, malignant melanoma of headand neck, lung cancer, non small cell lung cancer (NSCLC), squamous cellnon-small cell lung cancer, breast cancer, recurrent metastatic breastcancer, hepatocellular carcinoma, hodgkin's lymphoma, follicularlymphoma, non-hodgkin's lymphoma, advanced B-cell NHL, HL includingdiffuse large B-cell lymphoma (DLBCL), multiple myeloma, chronic myeloidleukemia, adult acute myeloid leukemia in remission; adult acute myeloidleukemia with Inv(16)(p13.1q22); CBFB-MYH11; adult acute myeloidleukemia with t(16;16)(p13.1;q22); CBFB-MYH11; adult acute myeloidleukemia with t(8;21)(q22;q22); RUNX1-RUNX1T1; adult acute myeloidleukemia with t(9;11)(p22;q23); MLLT3-MLL; adult acute promyelocyticleukemia with t(15;17)(q22;q12); PML-RARA; alkylating agent-relatedacute myeloid leukemia, chronic lymphocytic leukemia, richter'ssyndrome; waldenstrom macroglobulinemia, adult glioblastoma; adultgliosarcoma, recurrent glioblastoma, recurrent childhoodrhabdomyosarcoma, recurrent ewing sarcoma/peripheral primitiveneuroectodermal tumor, recurrent neuroblastoma; recurrent osteosarcoma,colorectal cancer, MSI positive colorectal cancer; MSI negativecolorectal cancer, nasopharyngeal nonkeratinizing carcinoma; recurrentnasopharyngeal undifferentiated carcinoma, cervical adenocarcinoma;cervical adenosquamous carcinoma; cervical squamous cell carcinoma;recurrent cervical carcinoma; stage IVA cervical cancer; stage IVBcervical cancer, anal canal squamous cell carcinoma; metastatic analcanal carcinoma; recurrent anal canal carcinoma, recurrent head and neckcancer; carcinoma, squamous cell of head and neck, head and necksquamous cell carcinoma (HNSCC), ovarian carcinoma, colon cancer,gastric cancer, advanced GI cancer, gastric adenocarcinoma;gastroesophageal junction adenocarcinoma, bone neoplasms, soft tissuesarcoma; bone sarcoma, thymic carcinoma, urothelial carcinoma, recurrentmerkel cell carcinoma; stage III merkel cell carcinoma; stage IV merkelcell carcinoma, myelodysplastic syndrome and recurrent mycosis fungoidesand Sezary syndrome.

Exemplary cancers that can be treated with the antibody combinationaccording to the present invention include cancers, such as lungadenocarcinoma, mucinous adenoma, ductal carcinoma of the pancreas andcolorectal carcinoma, brain lower grade glioma, breast invasivecarcinoma, glioblastoma multiforme, melanoma, thyroid, rectumadenocarcinoma, kidney cancer, renal cancer, liver cancer, acute myeloidleukemia, gastric adenocarcinoma, esophageal adenocarcinoma, uterinecorpus endometrioid carcinoma, bladder cancer, kidney cancer, prostatecancer, oral cancer, large intestine cancer and lymphoma.

It has been observed that many human tumors (deMartin et al., EMBO J.,6: 3673 (1987), Kuppner et al., Int. J. Cancer, 42: 562 (1988)) and manytumor cell lines (Derynck et al., Cancer Res., 47: 707 (1987), Robertset al., Br. J. Cancer, 57: 594 (1988)) produce TGFβ and suggests apossible mechanism for those tumors to evade normal immunologicalsurveillance.

TGFβ isoform expression in cancer is complex and variable with differentcombinations of TGFβ isoforms having different roles in particularcancers. TGFβ molecules can act both as tumor suppressors and tumorpromoters. For example, deletion or dowregulation of TGFβ signaling inanimals can result in increased breast cancer, intestinal cancer,pancreatic cancer, colon cancer and squamous cell carcinoma, indicatingthe presence of TGFβ is important to prevent or slow tumor progression(Yang et al., Trends Immunol 31:220-27, 2010). However, overexpressionof TGFβ is known to be pro-oncogenic and increased expression isdetected in many tumor types (Yang et al., supra)

Additional complexities are also disclosed in U.S. Pat. No. 7,927,593.For example, different TGFβ isoforms appear to be more relevant todifferent types of cancers. TGFβ1 and TGFβ3 may play a greater role inovarian cancer and its progression than TGβ2; while TGFβ1 and TGFβ2expression is greater in higher grade chondrosarcoma tumors than TGβ3.In human breast cancer, TGFβ1 and TGFβ3 are highly expressed, with TGFβ3expression correlating with overall survival, whereas patients with nodemetastasis and positive TGFβ3 expression have poor prognostic outcomes.However, in colon cancer, TGFβ1 and TGFβ2 are more highly expressed thanTGFβ3 and are present at greater circulating levels than in cancer-freeindividuals. In gliomas, TGFβ2 is important for cell migration.

Infiltration of immune cells into tumor sites is thought to be a commoncontributing factor to tumor growth. These immune cell infiltrates canhave a beneficial effect by helping to clear the tumor, but can also bedetrimental effect by enabling tolerance to tumor antigens. It has beenshown that TGFβ can affect levels of immune cells in tumors (see e.g.,Yang et al., Trends Immunol 31:220-27, 2010; Flavell et al., NatureImmunol 10:554-567, 2010; Nagarau et al., Expert Opin Investig Drugs19:77-91, 2010). For example, TGFβ suppresses natural killer cells thatinfiltrate tumors in order to clear tumors from the body. TGFβ alsosuppresses activity of cytotoxic T cells and CD4+ helper T cells, celltypes which assist in clearance of tumors (Yang, supra). TGFβ also playsa role in regulating dendritic cell activity, for example by inhibitingmigration into injury sites and presentation of antigen to promote animmune response. Dendritic cells are both responsive to TGFβ and secreteTGFβ. For example, dendritic cells infiltrate tumors and take up thecells, secrete TGFβ and activate regulatory T cells, which in turn canprevent tumor clearance (Flavell et al., supra). Additionally, myeloidderived suppressor cells (MDSC) are a bone marrow derived cells thatexpand during tumor progression. MDSC inhibit T cell proliferation,suppress dendritic cell maturation, and inhibit natural killer cellactivity, thereby helping cells to evade the immune response (Li et al.,J Immunol. 182:240-49, 2009). TGFβ has been demonstrated to contributeto the effects of MDSC on inhibiting natural killer cell activity (Li etal., supra; Xiang et al., Int J Cancer 124:2621-33, 2009). The role ofthe various TGFβ isoforms in each of these immune processes is unclear.Selectively targeting TGFβ isoforms and inhibiting them to varyingdegrees may be instrumental in modulating the host immune response tocombat and clear the tumor.

It is contemplated that the methods herein reduce tumor size or tumorburden in the subject, and/or reduce metastasis in the subject. Invarious embodiments, the methods reduce the tumor size by 10%, 20%, 30%or more. In various embodiments, the methods reduce tumor size by 10%,15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%,85%, 90%, 95% or 100%.

It is contemplated that the methods herein reduce tumor burden, and alsoreduce or prevent the recurrence of tumors once the cancer has gone intoremission.

In various embodiments, the TGFβ antibody and/or PD-1 antibody andcombinations thereof or compositions described herein modulates immunecells in a tumor. In some embodiments, the TGFβ antibody and/or PD-1antibody and combinations thereof or compositions herein increases thenumber of natural killer (NK) cells in a tumor and/or increasescytolytic activity of NK cells. In various embodiments, the antibodiesor compositions described herein decreases the number of regulatory Tcells in a tumor and/or inhibits regulatory T cell function. Forexample, in various embodiments, the antibodies or compositionsdescribed herein inhibits ability of Tregs to down-regulate an immuneresponse or to migrate to a site of an immune response.

In various embodiments, the TGFβ antibody and/or PD-1 antibody andcombinations thereof or compositions described herein increases thenumber of cytotoxic T cells in a tumor, and/or enhances CTL activity,e.g., boosts, increases or promotes CTL activity. For example, invarious embodiments, the antibodies or compositions described hereinincreases perforin and granzyme production by CTL and increasescytolytic activity of the CTL.

In various embodiments, the, the TGFβ antibody and/or PD-1 antibody andcombinations thereof or compositions described herein increases thenumber of dendritic cells (DC) in a tumor, and/or inhibits thetolerogenic function (e.g., tolerogenic effect) of dendritic cells. Forexample, in various embodiments, the antibodies or compositionsdescribed herein decreases the toleragenic effect of CD8+ dendriticcells.

In various embodiments, any of antibodies for TGFβ, XPA.42.068,XPA.42.089 or XPA.42.681 or variants thereof as described hereinmodulate one or more of the immune activities described above.

In one embodiment, treatment of cancer in an animal in need of saidtreatment, comprises administering to the animal an effective amount ofan inhibitor of TGFβ and an inhibitor of PD-1 or a compositioncomprising an inhibitor described herein. It is contemplated that theinhibitors are a TGFβ antibody and a PD-1 antibody.

The conditions treatable by methods of the present disclosure preferablyoccur in mammals. Mammals include, for example, humans and otherprimates, as well as pet or companion animals such as dogs and cats,laboratory animals such as rats, mice and rabbits, and farm animals suchas horses, pigs, sheep, and cattle.

Formulation of Pharmaceutical Compositions

To administer inhibitors, e.g., antibodies, of the present disclosure tohuman or test animals, it is preferable to formulate the inhibitors in acomposition comprising one or more pharmaceutically acceptable carriers.The phrase “pharmaceutically or pharmacologically acceptable” refer tomolecular entities and compositions that do not produce allergic, orother adverse reactions when administered using routes well-known in theart, as described below. “Pharmaceutically acceptable carriers” includeany and all clinically useful solvents, dispersion media, coatings,antibacterial and antifungal agents, isotonic and absorption delayingagents and the like.

In addition, compounds may form solvates with water or common organicsolvents. Such solvates are contemplated as well.

The inhibitors are administered by any suitable means, includingparenteral, subcutaneous, intraperitoneal, intrapulmonary, andintranasal, and, if desired for local treatment, intralesionaladministration. Parenteral infusions include intravenous, intraarterial,intraperitoneal, intramuscular, intradermal or subcutaneousadministration. In addition, the inhibitors are suitably administered bypulse infusion, particularly with declining doses of the antibody.Preferably the dosing is given by injections, most preferablyintravenous or subcutaneous injections, depending in part on whether theadministration is brief or chronic. Other administration methods arecontemplated, including topical, particularly transdermal, transmucosal,rectal, oral or local administration e.g. through a catheter placedclose to the desired site.

Pharmaceutical compositions of the present disclosure containing theinhibitors described herein as an active ingredient may containpharmaceutically acceptable carriers or additives depending on the routeof administration. Examples of such carriers or additives include water,a pharmaceutical acceptable organic solvent, collagen, polyvinylalcohol, polyvinylpyrrolidone, a carboxyvinyl polymer,carboxymethylcellulose sodium, polyacrylic sodium, sodium alginate,water-soluble dextran, carboxymethyl starch sodium, pectin, methylcellulose, ethyl cellulose, xanthan gum, gum Arabic, casein, gelatin,agar, diglycerin, glycerin, propylene glycol, polyethylene glycol,Vaseline, paraffin, stearyl alcohol, stearic acid, human serum albumin(HSA), mannitol, sorbitol, lactose, a pharmaceutically acceptablesurfactant and the like. Additives used are chosen from, but not limitedto, the above or combinations thereof, as appropriate, depending on thedosage form of the present disclosure.

Formulation of the pharmaceutical composition will vary according to theroute of administration selected (e.g., solution, emulsion). Anappropriate composition comprising the inhibitor, e.g., an antibody, tobe administered can be prepared in a physiologically acceptable vehicleor carrier. For solutions or emulsions, suitable carriers include, forexample, aqueous or alcoholic/aqueous solutions, emulsions orsuspensions, including saline and buffered media. Parenteral vehiclescan include sodium chloride solution, Ringer's dextrose, dextrose andsodium chloride, lactated Ringer's or fixed oils. Intravenous vehiclescan include various additives, preservatives, or fluid, nutrient orelectrolyte replenishers.

A variety of aqueous carriers, e.g., sterile phosphate buffered salinesolutions, bacteriostatic water, water, buffered water, 0.4% saline,0.3% glycine, and the like, and may include other proteins for enhancedstability, such as albumin, lipoprotein, globulin, etc., subjected tomild chemical modifications or the like.

Therapeutic formulations of the inhibitors are prepared for storage bymixing the inhibitor having the desired degree of purity with optionalphysiologically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions. Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.Such molecules are suitably present in combination in amounts that areeffective for the purpose intended.

The active ingredients may also be entrapped in microcapsule prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes.

Aqueous suspensions may contain the active compound in admixture withexcipients suitable for the manufacture of aqueous suspensions. Suchexcipients are suspending agents, for example sodiumcarboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia;dispersing or wetting agents may be a naturally-occurring phosphatide,for example lecithin, or condensation products of an alkylene oxide withfatty acids, for example polyoxyethylene stearate, or condensationproducts of ethylene oxide with long chain aliphatic alcohols, forexample heptadecaethyl-eneoxycetanol, or condensation products ofethylene oxide with partial esters derived from fatty acids and ahexitol such as polyoxyethylene sorbitol monooleate, or condensationproducts of ethylene oxide with partial esters derived from fatty acidsand hexitol anhydrides, for example polyethylene sorbitan monooleate.The aqueous suspensions may also contain one or more preservatives, forexample ethyl, or n-propyl, p-hydroxybenzoate.

The antibodies described herein can be lyophilized for storage andreconstituted in a suitable carrier prior to use. This technique hasbeen shown to be effective with conventional immunoglobulins. Anysuitable lyophilization and reconstitution techniques can be employed.It will be appreciated by those skilled in the art that lyophilizationand reconstitution can lead to varying degrees of antibody activity lossand that use levels may have to be adjusted to compensate.

The TGFβ and PD-1 antibodies described herein can be prepared andadministered as a co-formulation. In one aspect, at least two of theantibodies recognize and bind different antigens. In another aspect, atleast two of the plurality of antibodies can specifically recognize andbind different epitopes of the same antigen.

Dispersible powders and granules suitable for preparation of an aqueoussuspension by the addition of water provide the active compound inadmixture with a dispersing or wetting agent, suspending agent and oneor more preservatives. Suitable dispersing or wetting agents andsuspending agents are exemplified by those already mentioned above.

The concentration of antibody in these formulations can vary widely, forexample from less than about 0.5%, usually at or at least about 1% to asmuch as 15 or 20% by weight and will be selected primarily based onfluid volumes, viscosities, etc., in accordance with the particular modeof administration selected. Thus, a typical pharmaceutical compositionfor parenteral injection could be made up to contain 1 ml sterilebuffered water, and 50 mg of antibody. A typical composition forintravenous infusion could be made up to contain 250 ml of sterileRinger's solution, and 150 mg of antibody. Actual methods for preparingparenterally administrable compositions will be known or apparent tothose skilled in the art and are described in more detail in, forexample, Remington's Pharmaceutical Science, 15th ed., Mack PublishingCompany, Easton, Pa. (1980). An effective dosage of antibody is withinthe range of 0.01 mg to 1000 mg per kg of body weight peradministration.

The pharmaceutical compositions may be in the form of a sterileinjectable aqueous, oleaginous suspension, dispersions or sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersions. The suspension may be formulated according tothe known art using those suitable dispersing or wetting agents andsuspending agents which have been mentioned above. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example as a solution in 1,3-butane diol. The carrier can be asolvent or dispersion medium containing, for example, water, ethanol,polyol (for example, glycerol, propylene glycol, and liquid polyethyleneglycol, and the like), suitable mixtures thereof, vegetable oils,Ringer's solution and isotonic sodium chloride solution. In addition,sterile, fixed oils are conventionally employed as a solvent orsuspending medium. For this purpose any bland fixed oil may be employedincluding synthetic mono- or diglycerides. In addition, fatty acids suchas oleic acid find use in the preparation of injectables.

In all cases the form must be sterile and must be fluid to the extentthat easy syringability exists. The proper fluidity can be maintained,for example, by the use of a coating, such as lecithin, by themaintenance of the required particle size in the case of dispersion andby the use of surfactants. It must be stable under the conditions ofmanufacture and storage and must be preserved against the contaminatingaction of microorganisms, such as bacteria and fungi. The prevention ofthe action of microorganisms can be brought about by variousantibacterial and antifungal agents, for example, parabens,chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In manycases, it will be desirable to include isotonic agents, for example,sugars or sodium chloride. Prolonged absorption of the injectablecompositions can be brought about by the use in the compositions ofagents delaying absorption, for example, aluminum monostearate andgelatin.

Compositions useful for administration may be formulated with uptake orabsorption enhancers to increase their efficacy. Such enhancers includefor example, salicylate, glycocholate/linoleate, glycholate, aprotinin,bacitracin, SDS, caprate and the like. See, e.g., Fix (J. Pharm. Sci.,85:1282-1285 (1996)) and Oliyai and Stella (Ann. Rev. Pharmacol.Toxicol., 32:521-544 (1993)).

Antibody compositions contemplated for use to inhibit target activity,including binding of the target to its cognate receptor or ligand,target-mediated signaling, and the like. In particular, the compositionsexhibit inhibitory properties at concentrations that are substantiallyfree of side effects, and are therefore useful for extended treatmentprotocols. For example, co-administration of an antibody compositionwith another, more toxic, cytotoxic agent can achieve beneficialinhibition of a condition or disorder being treated, while effectivelyreducing the toxic side effects in the patient.

In addition, the properties of hydrophilicity and hydrophobicity of thecompositions contemplated for use in the present disclosure are wellbalanced, thereby enhancing their utility for both in vitro andespecially in vivo uses, while other compositions lacking such balanceare of substantially less utility. Specifically, compositionscontemplated for use in the disclosure have an appropriate degree ofsolubility in aqueous media which permits absorption and bioavailabilityin the body, while also having a degree of solubility in lipids whichpermits the compounds to traverse the cell membrane to a putative siteof action. Thus, antibody compositions contemplated are maximallyeffective when they can be delivered to the site of target antigenactivity.

Administration and Dosing

In one aspect, methods of the present disclosure include a step ofadministering a pharmaceutical composition. In certain embodiments, thepharmaceutical composition is a sterile composition.

Methods of the present disclosure are performed using anymedically-accepted means for introducing therapeutics directly orindirectly into a mammalian subject, including but not limited toinjections, oral ingestion, intranasal, topical, transdermal,parenteral, inhalation spray, vaginal, or rectal administration. Theterm parenteral as used herein includes subcutaneous, intravenous,intramuscular, and intracisternal injections, as well as catheter orinfusion techniques. Administration by, intradermal, intramammary,intraperitoneal, intrathecal, retrobulbar, intrapulmonary injection andor surgical implantation at a particular site is contemplated as well.

In one embodiment, administration is performed at the site of a canceror affected tissue needing treatment by direct injection into the siteor via a sustained delivery or sustained release mechanism, which candeliver the formulation internally. For example, biodegradablemicrospheres or capsules or other biodegradable polymer configurationscapable of sustained delivery of a composition (e.g., a solublepolypeptide, antibody, or small molecule) can be included in theformulations of the disclosure implanted near or at site of the cancer.

Therapeutic compositions may also be delivered to the patient atmultiple sites. The multiple administrations may be renderedsimultaneously or may be administered over a period of time. In certaincases it is beneficial to provide a continuous flow of the therapeuticcomposition. Additional therapy may be administered on a period basis,for example, hourly, daily, every other day, twice weekly, three timesweekly, weekly, every 2 weeks, every 3 weeks, monthly, or at a longerinterval.

Also contemplated in the present disclosure is the administration ofmultiple agents, such as the antibody compositions in conjunction with athird agent as described herein, including but not limited to achemotherapeutic agent.

The amounts of inhibitor or antibody composition in a given dosage mayvary according to the size of the individual to whom the therapy isbeing administered as well as the characteristics of the disorder beingtreated. In exemplary treatments, it may be necessary to administerabout 1 mg/day, 5 mg/day, 10 mg/day, 20 mg/day, 50 mg/day, 75 mg/day,100 mg/day, 150 mg/day, 200 mg/day, 250 mg/day, 500 mg/day or 1000mg/day. These concentrations may be administered as a single dosage formor as multiple doses. Standard dose-response studies, first in animalmodels and then in clinical testing, reveals optimal dosages forparticular disease states and patient populations.

Also contemplated in the present disclosure, the amounts of TGFβinhibitor and PD-1 inhibitor in a given dosage may vary according to thesize of the individual to whom the therapy is being administered as wellas the characteristics of the disorder being treated. Both inhibitorcompositions can be administered in a dose range of 0.1 to 15 mg as anintravenous infusion over 30-60 minutes every 1-4 weeks until diseaseprogression or unacceptable toxicity. In various embodies the dose canbe 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 mg/kg.

It will also be apparent that dosing may be modified if traditionaltherapeutics are administered in combination with therapeutics of thedisclosure.

Kits

As an additional aspect, the disclosure includes kits which comprise oneor more compounds or compositions packaged in a manner which facilitatestheir use to practice methods of the disclosure. In one embodiment, sucha kit includes a compound or composition described herein (e.g., acomposition comprising a target-specific antibody alone or incombination with another antibody or a third agent), packaged in acontainer such as a sealed bottle or vessel, with a label affixed to thecontainer or included in the package that describes use of the compoundor composition in practicing the method. Preferably, the compound orcomposition is packaged in a unit dosage form. The kit may furtherinclude a device suitable for administering the composition according toa specific route of administration or for practicing a screening assay.Preferably, the kit contains a label that describes use of the inhibitorcompositions.

Additional aspects and details of the disclosure will be apparent fromthe following examples, which are intended to be illustrative ratherthan limiting.

EXAMPLES Example 1. TGFβ and PD-1 Inhibitor Combination Therapy ofChemically Induced Cutaneous Squamous Cell Carcinoma (cSCC)

To demonstrate the effect of TGFβ and PD-1 inhibitor combinationtherapy, FVB mice were subcutaneously injected with chemically-inducedKrasG13C-driven cSCC tumor line 168 (cultured ≤2 passages in vitro).When tumors reached ˜3-5 mm diameter (approximately 2-3 weekspost-implantation), mice were treated with either anti-PD-1 (α-PD-1)antibody alone (250 μg, Clone: RMP1-14, Cat. no.: BE0146, BioXCell),pan-specific α-TGFβ1, 2, 3 (anti-TGFβ) antibody (200 μg) alone or α-PD-1and pan-specific α-TGFβ1, 2, 3 antibodies combined via intraperitonealinjection (i.p.), three treatments at 4 day intervals (mice injected onday 0, day 4 and day 8, n=7 per arm). Tumor sizes were subsequentlymeasured using a caliper. α-PD-1 monotherapy inhibited tumor growthcompared with control mice but the tumor regression was not sustainedafter 12-14 days. Unlike α-PD-1 monotherapy, α-TGFβ monotherapy showedrobust and sustained activity (>12-14 days). While both α-PD-1 andα-TGFβ mono-therapy induced tumor regression compared to matched IgGcontrol-treated mice, the combined effect of α-PD-1 and α-TGFβ onaverage was greater than that of either reagent alone (FIG. 1A, *P<0.05,**P<0.01, ***P <0.0001). Tumors were separated into α-PD-1 progressorsor resistant (‘res’), α-PD-1 responders or sensitive (‘sens’), α-TGFβresponders (TGFb) and α-PD-1/α-TGFβ combination responders (PD-1 TGFb).Levels of immune cell markers in these tumors were measured as apercentage of live cells and as a percentage of CD45+ cells to assesschanges in populations of tumor infiltrating immune cells in response todrug. CD45+, Natural killer (NK) cells, regulatory T (Treg) cells, CD4+and CD8+ T cells were measured by Fluorescence-activated cell sorting(FACS) in 2-3 tumors per cohort at day 8, following the third treatmentwith inhibitors (FIGS. 1B and 1C). Elevated CD8+ cell numbers wereutilized as a biomarker of α-TGFβ and α-PD-1 inhibitor responsiveness.

Example 2. Differential Responses of Individual Tumors to Immunotherapy

Data from FIG. 1 were segregated into “responders” and “progressors” todemonstrate the range of responses to α-PD-1 and/or α-TGFβ immunotherapy(FIG. 2). In the case of α-TGFβ monotherapy and α-PD1 and α-TGFβcombinational therapy, regression of the implanted tumor was observed in3 out of the 7 treated mice. Furthermore with the combined α-PD1 andα-TGFβ treatment, complete tumor regression was observed in ˜50% cases,with no further tumor outgrowth four weeks post-dosing with noadditional drug doses (i.e., a sustained reduction in tumor size).

These results show that the combination of α-TGFβ antibody and α-PD-1antibody is more effective than single antibody treatment at promotingtumor regression, and surprisingly, at preventing the recurrence ofcancer.

Example 3. Reduction in Tumor Size in with α-TGFβ/α-PD-1 CombinationalTherapy in Chemically-Induced (DMBA-TPA) Cutaneous Squamous CellCarcinoma (cSCC) Mice

In addition to the use of allograft models, the anti-tumor effects ofpan-specific α-TGFβ1, 2, 3 α-TGFβ/α-PD-1 combinational therapy were alsoassessed using a mouse skin model of direct chemically-inducedcarcinogenesis. The DMBA-TPA (12-Otetradecanoyl-phorbol-13-acetate)induced cSCCs model has been well characterized (Balmain A et al.,Princess Takamatsu Symp., 22:97-108, 1991; Burns P A et al., Oncogene,6(12):2363-9, 1991; Yuspa S H et al., Dermatol Symp Proc., 1(2):147-50,1996; Frame S et al., Philos Trans R Soc Lond B Biol Sci.353(1370):839-45, 1998) and used extensively to identify genetic andmolecular mechanisms of cancer initiation and progression that would nothave been attainable from studying human cancer or more simple models,such as tumor allografts. The model is initiated by DMBA-mutation ofHras codon 61 in >90% of tumors in wild type mice (Quintanilla M et al.,Nature., 322(6074): 78-80, 1986). Subsequent multiple biweeklytreatments with the tumor promoter, TPA, stimulate the outgrowth ofbenign premalignant papillomas. A proportion of papillomas progress tocarcinomas over a period of 4-12 months, and in some cases to highlyinvasive spindle tumors that have lost many of the characteristics oftheir epithelial cell.

Using a bioluminescence reporter strain of luciferase knockin mice(p16(LUC)), the growth of chemically-induced cSCCs was measuredpreceding and then after treatment of the mice with a combination ofα-PD1 and α-TGFβ antibodies. p16-LUC mice report the expression ofp16(INK4a) gene, a tumor suppressor in Ras-driven tumor cells. Thereporter is activated by early neoplastic events, enabling visualizationof tumors and measurement of tumor size using a IVIS optical imagingsystem.

Tumors were initiated by topically treating 3 mice with DMBA twice inthe first week and TPA for the following 20 weeks with carcinomaoutgrowth observed at ˜30 weeks. Surgical resection of carcinomas wasperformed at 32 weeks after tumor initiation with DMBA treatment. Micewere imaged and tumor sizes were measured 5 times (Pre-surgicalresection: at week 0 and post-surgical resection: at weeks 8, 12, 21 and27).

When all mice had approximately 15 mm carcinomas and small lungmetastases (3×3 mm), carcinomas were resectioned and mice were treatedwith α-PD-1 antibody (250 μg, Clone: RMP1-14, BioXCell) and α-TGFβantibody (200 μg) combined via intraperitoneal injection (i.p.), threetreatments at 4 day intervals (mice injected on day 8, day 12 and day16, n=3). Tumor sizes were subsequently measured using a luminescenceimager. Combinational α-PD-1 and α-TGFβ antibody treatments resulted intumor regression in all 3 mice.

Carcinomas that were resectioned from mice were immunotyped for cellmarkers CD45+, Natural killer (NK) cells, regulatory T (Treg) cells,CD4+ and CD8+ T cells were measured by FACS in tumors following thethird treatment with inhibitors. Tumors were separated into α-PD-1progressors, α-PD-1 responders, α-TGFβ responders and α-PD-1/α-TGFβcombination responders. Levels of immune cell markers in these tumorswere measured as a percentage of live cells and as a percentage of CD45+cells to assess changes in populations of tumor infiltrating immunecells in response to drug (FIG. 4). Tumors which responded to treatmentwith α-PD-1 alone, α-TGFβ alone and α-PD-1/α-TGFβ in combination showedsignificantly increased levels of CD4+ and CD8+ T cells, withα-PD-1/α-TGFβ in combination responsive tumors showing even higherlevels of CD4+ T cell subsets. In addition, α-TGFβ alone andα-PD-1/α-TGFβ in combination responsive tumors showed CD8+T effector(Teff)/Tregulator (Treg) CD45+ cells. Elevated CD8+ T cell numbers wereutilized as a biomarker of α-TGFβ and α-PD-1 inhibitor responsiveness.

Example 4. Chemically Induced Kras-Driven SCC Tumors with the GreatestMutational Load Respond to α-PD1 and α-TGFβ Monotherapy andCombinational Therapy

TGFβ and PD-1 inhibitor monotherapy and combinational therapy wereevaluated using chemically-induced (DMBA/TPA) Kras- and Hras-drivenversus genetically-initiated (GEMM) Kras-driven cSCC cell lines insyngeneic FVB/N mice. Chemically-induced tumor cell lines often possessmore mutations than genetically induced cell lines. These highermutation numbers, are more similar to the number of mutations in humantumors (Westcott P W et al., Nature, 517: 489-92, 2015). The followingchemically-induced cell lines: FVB-62, FVB-85, FVB-166, FVB-168, FVB-169were compared with the following Kras-driven GEMM-initiated models ofcSCC: FVB-1425, FVB-1428, using the methods described in example 1,para. [281] with mice treated twice with α-PD1 monotherapy, α-TGFβmonotherapy or α-PD-1 and α-TGFβ antibodies combined (or controlantibodies) once on day 0 and once on day 4 followed by tumorimmunotyping on days 6-10. Results of these experiments showed that onlychemically-induced Kras-driven SCC tumors reported to harbor thegreatest mutational load (i.e. greater number of mutations per MB, whichare more representative of human tumors) responded to pan-specificα-TGFβ1, 2, 3 and α-PD1 monotherapy and combinational therapy (FIG. 5).The treatment sensitive, chemically induced SCC tumor cell lines wereFVB-168 and FVB-169 with KrasG13R mutations.

Using the chemically-induced SCC tumor cell line FVB-168, response topan specific α-TGFβ1, 2, 3 and α-PD1 monotherapy and combinationaltherapy was evaluated by measuring the reduction in carcinomas andexpressed as a partial response, a complete response or progressivedisease (FIG. 6A, data from two independent experiments). Pan-specificα-TGFβ1, 2, 3 and α-PD1 monotherapy inhibited disease progression by˜40-50%. No significant difference in disease was observed betweeneither therapy alone. However, pan-specific α-TGFβ1, 2, 3 and α-PD1combinational therapy was found to be significantly more effective atinhibiting disease progression compared with either pan-specificα-TGFβ1, 2, 3 or α-PD1 monotherapy. Pan-specific α-TGFβ1, 2, 3 and α-PD1combinational therapy resulted in a ˜90% reduction in diseaseprogression compared with control animals and was associated with betteroverall survival (FIG. 6B).

Tumors were separated into progressing carcinomas (i.e., tumor(s)continuing to grow) or responding carcinomas (i.e., tumor growthinhibited by therapy). Levels of immune cell markers in these tumorswere measured as a percentage of CD45+ cells to assess changes inpopulations of tumor infiltrating immune cells in response to drug. CD8+effector (T_(eff)), CD4+ effector (T_(eff)), CD4+ regulatory T (T_(reg))cells and the ratio of T_(eff)/T_(reg) were measured byFluorescence-activated cell sorting (FACS) in tumors per cohort betweendays 6-10 following treatment with α-PD1 monotherapy, pan-specificα-TGFβ1, 2, 3 monotherapy or α-PD-1 and pan-specific α-TGFβ1, 2, 3combined (or control antibodies). α-TGFβ and α-PD-1 inhibition resultedin expansion of CD8+ and CD4+T effector cells (FIG. 6C).

These results show that tumors that have characteristics similar tohuman tumors are highly responsive to treatment with a combination ofTGFβ and PD-1 inhibitors and that the combination therapy reducesoverall progression of disease. TGFβ/PD-1 inhibitor combination therapyalso changes the immunological profile of cells in the tumors to a morefavorable effector T cell to regulatory T cell ratio (Teff/Treg).

Example 5. Comparison of TGFβ1, 2 Specific and Pan-Specific TGFβ1, 2, 3Antibodies and α-PD-1 Combination Therapy in Allograft Tumor Model

In order to compare the effects of antibodies that block TGFβ1, 2 and 3with those that block only TGFβ1, 2, either as a monotherapy or in PD-1inhibitor combination therapy, pan-specific α-TGFβ1, 2, 3 and TGFβ1, 2specific antibodies were assayed in an allograft model of Kras-drivenSCC.

FVB mice (n=70) were subcutaneously injected with the cSCC tumor line,Hras168 (1.25×10⁴ cells). Once tumors were palpable (˜0.5 cm diameter,˜2 weeks post-implantation), mice were sorted into six experimental arms(n=9-10 per arm) containing mice of similar body weight and tumor size.Mice were treated with three consecutive doses at day 0, day 4 and day8, with 200 μg per dose of either pan-specific α-TGFβ1, 2, 3 or α-TGFβ1,2 and/or 250 μg α-PD-1 per dose and/or matched control antibodies at thesame concentrations. The six experimental arms included: 1) controlantibodies; 2) α-PD1; 3) α-TGFβ1, 2 specific; 4) α-TGFβ1, 2 specific andα-PD-1 combined; 5) pan-specific α-TGFβ1, 2, 3; 6) pan-specific α-TGFβ1,2, 3 antibody and α-PD-1 combined via intraperitoneal injection (i.p.).Tumor sizes were subsequently measured using a caliper. Bothpan-specific α-TGFβ1, 2, 3 and α-TGFβ1, 2 monotherapies inhibited tumorgrowth (i.e., reduced tumor size) compared to controls and were bothmore efficacious than the α-PD-1 antibody alone. Furthermore, bothpan-specific α-TGFβ1, 2, 3 and α-TGFβ1, 2 antibodies showed comparableanti-tumor activity to each other in both monotherapy and in combinationtherapy with α-PD-1 (FIG. 7).

These results show that an antibody that can block either TGFβ1, 2, 3isoforms or TGFβ1, 2 isoforms is effective in combination with a PD-1inhibitor in reducing tumor burden in a subject.

Numerous modifications and variations in the disclosure as set forth inthe above illustrative examples are expected to occur to those skilledin the art. Consequently only such limitations as appear in the appendedclaims should be placed on the disclosure.

What is claimed:
 1. A method for treating cancer or preventing therecurrence of cancer comprising administering to a subject in needthereof therapeutically effective amounts of an inhibitor oftransforming growth factor beta (TGFβ) and an inhibitor of Programmedcell death protein 1 (PD-1).
 2. The method of claim 1, wherein the TGFβinhibitor is an antibody that binds TGFβ1, TGFβ2 and TGFβ3.
 3. Themethod of claim 1, wherein the TGFβ inhibitor is an antibody that bindsto TGFβ1, TGFβ2 with greater affinity than to TGFβ3.
 4. The method ofclaim 1, wherein the TGFβ inhibitor neutralizes activity of TGFβ1 andTGF β2 to a greater extent than it neutralizes activity of TGF β3. 5.The method of claim 2, wherein the TGFβ antibody binds to TGFβ1, TGFβ2and TGFβ3 with an affinity Kd of 10⁻⁶ M or less.
 6. The method of anyone of the preceding claims wherein the TGFβ inhibitor is an antibodycomprising: (a) a heavy chain CDR1 amino acid sequence set forth in SEQID NOs: 13, 19 and 25, or a variant thereof having at least 85% identitythereto; (b) a heavy chain CDR2 amino acid sequence set forth in SEQ IDNOs: 14, 20 and 26, or a variant thereof having at least 85% identitythereto; (c) a heavy chain CDR3 amino acid sequence set forth in SEQ IDNOs: 15, 21 and 27, or a variant thereof having at least 85% identitythereto; (d) a light chain CDR1 amino acid sequence set forth in SEQ IDNOs: 16, 22 and 28, or a variant thereof having at least 85% identitythereto; (e) a light chain CDR2 amino acid sequence set forth in SEQ IDNOs: 17, 23 and 29, or a variant thereof having at least 85% identitythereto; and (f) a light chain CDR3 amino acid sequence set forth in SEQID NOs: 18, 24 and 30, or a variant thereof having at least 85% identitythereto.
 7. The method of any one of the preceding claims wherein theTGFβ inhibitor is an antibody that comprises an amino acid sequence atleast 85% identical to a heavy chain variable region amino acid sequenceset forth in SEQ ID NOs: 2, 6 and
 10. 8. The method of claim 7 whereinthe antibody further comprises an amino acid sequence at least 85%identical to a light chain variable region amino acid sequence set forthin SEQ ID NOs: 4, 8 and
 12. 9. The method of any one of the precedingclaims wherein the TGFβ inhibitor is an antibody comprising: a) a heavychain CDR1 amino acid sequence set forth in SEQ ID NO: 25, or a variantthereof in which one or two amino acids have been changed; b) a heavychain CDR2 amino acid sequence set forth in SEQ ID NO: 26, or a variantthereof in which one or two amino acids have been changed; c) a heavychain CDR3 amino acid sequence set forth in SEQ ID NO: 27, or a variantthereof in which one or two amino acids have been changed; d) a lightchain CDR1 amino acid sequence set forth in SEQ ID NO: 28, or a variantthereof in which one or two amino acids have been changed; e) a lightchain CDR2 amino acid sequence set forth in SEQ ID NO: 29, or a variantthereof in which one or two amino acids have been changed; and f) alight chain CDR3 amino acid sequence set forth in SEQ ID NO: 30, or avariant thereof in which one or two amino acids have been changed. 10.The method of any one of the preceding claims wherein the TGFβ inhibitoris an antibody comprising: a) a heavy chain CDR1 amino acid sequence setforth in SEQ ID NO: 13, or a variant thereof in which one or two aminoacids have been changed; b) a heavy chain CDR2 amino acid sequence setforth in SEQ ID NO: 14, or a variant thereof in which one or two aminoacids have been changed; c) a heavy chain CDR3 amino acid sequence setforth in SEQ ID NO: 15, or a variant thereof in which one or two aminoacids have been changed; d) a light chain CDR1 amino acid sequence setforth in SEQ ID NO: 16, or a variant thereof in which one or two aminoacids have been changed; e) a light chain CDR2 amino acid sequence setforth in SEQ ID NO: 17, or a variant thereof in which one or two aminoacids have been changed; and f) a light chain CDR3 amino acid sequenceset forth in SEQ ID NO: 18, or a variant thereof in which one or twoamino acids have been changed.
 11. The method of any one of thepreceding claims wherein the TGFβ inhibitor is an antibody comprising:g) a heavy chain CDR1 amino acid sequence set forth in SEQ ID NO: 19, ora variant thereof in which one or two amino acids have been changed; h)a heavy chain CDR2 amino acid sequence set forth in SEQ ID NO: 20, or avariant thereof in which one or two amino acids have been changed; i) aheavy chain CDR3 amino acid sequence set forth in SEQ ID NO: 21, or avariant thereof in which one or two amino acids have been changed; j) alight chain CDR1 amino acid sequence set forth in SEQ ID NO: 22, or avariant thereof in which one or two amino acids have been changed; k) alight chain CDR2 amino acid sequence set forth in SEQ ID NO: 23, or avariant thereof in which one or two amino acids have been changed;and 1) a light chain CDR3 amino acid sequence set forth in SEQ ID NO:24, or a variant thereof in which one or two amino acids have beenchanged.
 12. The method of claim 5 wherein the heavy chain variableregion amino acid sequence is set forth in SEQ ID NO: 10 and the lightchain variable region amino acid sequence is set forth in SEQ ID NO: 12.13. The method of claim 5 wherein the heavy chain variable region aminoacid sequence is set forth in SEQ ID NO: 2 and the light chain variableregion amino acid sequence is set forth in SEQ ID NO:
 4. 14. The methodof claim 5 wherein the heavy chain variable region amino acid sequenceis set forth in SEQ ID NO: 6 and the light chain variable region aminoacid sequence is set forth in SEQ ID NO:
 8. 15. The method of any one ofclaims 6 to 14 wherein the antibody further comprises a heavy chainconstant region, wherein the heavy chain constant region is a modifiedor unmodified IgG, IgM, IgA, IgD, IgE, a fragment thereof, orcombinations thereof.
 16. The method of claim 15 further comprising ahuman light chain constant region attached to said light chain variableregion.
 17. The method of claim 1, wherein the PD-1 inhibitor is anantibody that binds PD-1.
 18. The method of claim 1 wherein the TGFβinhibitor is an antibody and the PD-1 inhibitor is an antibody.
 19. Themethod of claim 1, wherein the cancer is selected from the groupconsisting of esophageal cancer, pancreatic cancer, metastaticpancreatic cancer, metastatic adenocarcinoma of the pancreas, bladdercancer, stomach cancer, fibrotic cancer, glioma, malignant glioma,diffuse intrinsic pontine glioma, recurrent childhood brain neoplasmrenal cell carcinoma, clear-cell metastatic renal cell carcinoma, kidneycancer, prostate cancer, metastatic castration resistant prostatecancer, stage IV prostate cancer, metastatic melanoma, melanoma,malignant melanoma, recurrent melanoma of the skin, melanoma brainmetastases, stage IIIA skin melanoma; stage IIIB skin melanoma, stageIIIC skin melanoma; stage IV skin melanoma, malignant melanoma of headand neck, lung cancer, non small cell lung cancer (NSCLC), squamous cellnon-small cell lung cancer, breast cancer, recurrent metastatic breastcancer, hepatocellular carcinoma, hodgkin's lymphoma, follicularlymphoma, non-hodgkin's lymphoma, advanced B-cell NHL, HL includingdiffuse large B-cell lymphoma (DLBCL), multiple myeloma, chronic myeloidleukemia, adult acute myeloid leukemia in remission; adult acute myeloidleukemia with Inv(16)(p13.1q22); CBFB-MYH11; adult acute myeloidleukemia with t(16;16)(p13.1;q22); CBFB-MYH11; adult acute myeloidleukemia with t(8;21)(q22;q22); RUNX1-RUNX1T1; adult acute myeloidleukemia with t(9;11)(p22;q23); MLLT3-MLL; adult acute promyelocyticleukemia with t(15;17)(q22;q12); PML-RARA; alkylating agent-relatedacute myeloid leukemia, chronic lymphocytic leukemia, richter'ssyndrome; waldenstrom macroglobulinemia, adult glioblastoma; adultgliosarcoma, recurrent glioblastoma, recurrent childhoodrhabdomyosarcoma, recurrent ewing sarcoma/peripheral primitiveneuroectodermal tumor, recurrent neuroblastoma; recurrent osteosarcoma,colorectal cancer, MSI positive colorectal cancer; MSI negativecolorectal cancer, nasopharyngeal nonkeratinizing carcinoma; recurrentnasopharyngeal undifferentiated carcinoma, cervical adenocarcinoma;cervical adenosquamous carcinoma; cervical squamous cell carcinoma;recurrent cervical carcinoma; stage IVA cervical cancer; stage IVBcervical cancer, anal canal squamous cell carcinoma; metastatic analcanal carcinoma; recurrent anal canal carcinoma, recurrent head and neckcancer; carcinoma, squamous cell of head and neck, head and necksquamous cell carcinoma (HNSCC), ovarian carcinoma, colon cancer,gastric cancer, advanced GI cancer, gastric adenocarcinoma;gastroesophageal junction adenocarcinoma, bone neoplasms, soft tissuesarcoma; bone sarcoma, thymic carcinoma, urothelial carcinoma, recurrentmerkel cell carcinoma; stage III merkel cell carcinoma; stage IV merkelcell carcinoma, myelodysplastic syndrome and recurrent mycosis fungoidesand Sezary syndrome.
 20. The method of any one of the preceding claimswherein the cancer is selected from the group consisting of non smallcell lung carcinoma (NSCLC), head and neck cancer, skin cancer, melanomaand squamous cell carcinoma (SCC).
 21. The method of any one of thepreceding claims wherein the cancer has a mutation in the V-Ki-ras2Kirsten rat sarcoma viral oncogene homolog (KRAS) oncogene.
 22. Themethod of any one of the preceding claims wherein the cancer has amutation in the Harvey rat sarcoma viral oncogene homolog (HRAS)oncogene.
 23. The method of any one of the preceding claims wherein thecancer has a mutation in the neuroblastoma RAS viral (v-ras) oncogenehomolog (NRAS) oncogene.
 24. The method of any one of the precedingclaims wherein the cancer has mutations in the RAS oncogene.
 25. Themethod of any one of claims 21-24, wherein the cancer is selected fromthe group consisting of lung adenocarcinoma, mucinous adenoma, ductalcarcinoma of the pancreas and colorectal carcinoma, brain lower gradeglioma, breast invasive carcinoma, glioblastoma multiforme, melanoma,thyroid, rectum adenocarcinoma, kidney cancer, renal cancer, livercancer, acute myeloid leukemia, gastric adenocarcinoma, esophagealadenocarcinoma, uterine corpus endometrioid carcinoma, bladder cancer,kidney cancer, prostate cancer, oral cancer, large intestine cancer andlymphoma.
 26. The method of any one of the preceding claims comprisingreducing tumor size or tumor burden in the subject.
 27. The method ofany one of the preceding claims comprising reducing metastasis in thesubject.
 28. The method of any one of the preceding claims wherein thetumor size is reduced by 20% or more.
 29. The method of any one of thepreceding claims wherein the PD-1 inhibitor and the TGFβ inhibitor areformulated in a pharmaceutical composition.
 30. The method of claim 29wherein the inhibitors are in the same composition.
 31. The method ofclaim 29 wherein the inhibitors are in separate compositions.
 32. Themethod of any one of the preceding claims wherein the inhibitors areadministered concurrently.
 33. The method of any one of the precedingclaims wherein the inhibitors are administered at separate times orconsecutively.
 34. The method of any one of the preceding claims whereinthe administration prevents the recurrence of cancer in a subject thathas received inhibitor therapy.
 35. The method of any one of thepreceding claims, wherein the administration increases the number ofnatural killer (NK) cells in a tumor and/or improves NK cell cytolyticactivity.
 36. The method of any one of the preceding claims, wherein theadministration decreases the number of regulatory T cells in a tumorand/or inhibits regulatory T cell function.
 37. The method of any one ofthe preceding claims, wherein administration increases the number ofcytotoxic T cells (CTLs) in a tumor and/or enhances CTL function. 38.The method of any one of the preceding claims, wherein theadministration increases the number of type 2 dendritic cells (DC2) in atumor.
 39. The method of any of the preceding claims wherein theinhibitors are administered once daily, once weekly, twice weekly, onceevery two weeks, once every three weeks, monthly or once every twomonths.
 40. The method of any of the preceding claims wherein the TGFβinhibitor is administered in a dose range of 0.1 to 15 mg/kg and thePD-1 inhibitor is administered in a dose range from 0.1 to 15 mg/kg. 41.The method of any one of the preceding claims, wherein theadministration increases the ratio of effector T cells to regulatory Tcells in a tumor.
 42. A method for increasing the ratio of effector Tcells to regulatory T cells in a tumor comprising administering to asubject in need thereof therapeutically effective amounts of aninhibitor of transforming growth factor beta (TGFβ) and an inhibitor ofProgrammed cell death protein 1 (PD-1).